Preface

Preface

The United States federal animal welfare regulations and the Public Health Service Policy on Humane Care and Use of Laboratory Animals require that institutional animal care and use committees (IACUCs) conduct continuing reviews of all animal research activities. However, little is known about the lapse rate of IACUC continuing reviews, and how frequently investigators continue research activities during the lapse. It is also not clear what factors may contribute to an institution’s lapse in IACUC continuing reviews. As part of the quality assurance program, the Department of Veterans Affairs (VA) has collected performance metric data for animal care and use programs since 2011. We analyzed IACUC continuing review performance data at 74–75 VA research facilities from 2011 through 2015. The IACUC continuing review lapse rates improved from 5.6% in 2011 to 2.7% in 2015. The rate of investigators continuing research activities during the lapse also decreased from 47.2% in 2012 to 7.4% in 2015. The type of IACUCs used and the size of animal research programs appeared to have no effect in facility’s rates of lapse in IACUC continuing reviews. While approximately 80% of facilities reported no lapse in IACUC continuing reviews, approximately 14% of facilities had lapse rates of >10% each year. Some facilities appeared to be repeat offenders. Four facilities had IACUC lapse rates of >10% in at least 3 out of 5 years, suggesting a system problem in these facilities requiring remedial actions to improve their IACUC continuing review processes.

The review articles in this issue describe animal models (predominantly rodents) that have contributed to monumental progress and advancement of knowledge in the fi eld of addictive and neurobiological behavior research (ANBR 1 ). This article provides brief guidance to institutional animal care and use committee (IACUC) members for their review of ANBR protocols involving animal models, with an emphasis on rodent models. In addition to the 2011 Guide for the Care and Use of Laboratory Animals (NRC 2011; the Guide), researchers and IACUC members must ensure compliance with the requirements of the Public Health Service Policy (implemented under the NIH Offi ce of Laboratory Animal Welfare), the United States Department of Agriculture (USDA) Animal Welfare Act, and the Association for the Accreditation and Advancement of Laboratory Animal Care (AAALAC) International.

Cell Bio conferences-organized jointly by the American Society of Cell Biology (ASCB) and European Molecular Biology Organization (EMBO)-showcase a diverse global community of the brightest researchers in Cell Biology and in emerging interdisciplinary topics, including bioelectricity. In this report, we briefly overview the Cell Bio 2023 subgroup meeting "Bioelectricity in Development, Regeneration, and Cancers." This subgroup meeting featured 12 talks (7 Principal Investigators and 5 junior scientists) exploring the role of bioelectricity in endogenous and diseased states in model systems ranging from cells in culture to single-cell organisms such as yeast all the way to mammalian systems (including tools and technology developed for exploring bioelectricity and electrotaxis in cells and tissues). The subgroup meeting concluded with a discussion on the current challenges and opportunities for the field of bioelectricity.

BackgroundActivation of T cell responses is essential for effective tumor clearance, however generating targeted, effective antigen presentation to stimulate T cell response remains challenging. We can harness the natural process...

Cairo University was the first academic institution in Egypt to establish an Institutional Animal Care and Use Committee (IACUC), as mandated by the World Organisation for Animal Health (OIE). Animal-based research should be performed in accordance with international regulations to monitor the humane care and use of the laboratory animals. Until 2018, the formal training of researchers in the appropriate and correct methods of animal handling during sampling and administration, as well as their husbandry demands, was an uncommon practice in Egypt. In 2018, the Egyptian Association for Animal Research Advancement (EAARA) organised the first international course in laboratory animal science (LAS), in collaboration with Utrecht University (The Netherlands) and the Faculty of Science, Cairo University, to raise researchers' awareness and increase their knowledge of the principles that govern the humane use and care of laboratory animals. A total of 26 researchers from a number of fields (veterinary medicine, dentistry, science, medicine, pharmacy and agriculture) enrolled in the course. In the responses to the post-course questionnaire, 24 (92.3%) participants stated that the principles of animal welfare (Three Rs) were well explained. In addition, 18 (69%) participants found that the course improved their skills in animal sampling and handling. Of the 26 participants, 22 (84.6%) became aware of their responsibility towards their experimental animals and agreed that the different methods of euthanasia were well explained. In conclusion, the general assessment of the course revealed a positive outcome regarding the culture of animal care; the course was repeated a year later, and several participants were enlisted as trainers in this second course.

It’s been 50 years since Women in Cell Biology (WICB) was founded by junior women cell biologists who found themselves neither represented at the American Society for Cell Biology (ASCB) presentations nor receiving the information, mentoring, and sponsorship they needed to advance their careers. Since then, gender parity at ASCB has made significant strides: WICB has become a standing ASCB committee, women are regularly elected president of the ASCB, and half the symposia speakers are women. Many of WICB’s pioneering initiatives for professional development, including career panels, workshops, awards for accomplishments in science and mentoring, and career mentoring roundtables, have been incorporated and adapted into broader “professional development” that benefits all members of ASCB. The time has passed when we can assume that all women benefit equally from progress. By strategically, thoughtfully, and honestly recognizing the challenges to women of the past and today, we may anticipate those new challenges that will arise in the next 50 years. WICB, in collaboration with the ASCB, can lead in data collection and access and can promote diversity, equity, and inclusion. This work will be a fitting homage to the women who, half a century ago, posted bathroom stall invitations to the first Women in Cell Biology meetup.

With the increasing affordability and accessibility of genome sequencing data, de novo genome assembly is an important first step to a wide variety of downstream studies and analyses. Therefore, bioinformatics tools that enable the generation of high-quality genome assemblies in a computationally efficient manner are essential. Recent developments in long-read sequencing technologies have greatly benefited genome assembly work, including scaffolding, by providing long-range evidence that can aid in resolving the challenging repetitive regions of complex genomes. ntLink is a flexible and resource-efficient genome scaffolding tool that utilizes long-read sequencing data to improve upon draft genome assemblies built from any sequencing technologies, including the same long reads. Instead of using read alignments to identify candidate joins, ntLink utilizes minimizer-based mappings to infer how input sequences should be ordered and oriented into scaffolds. Recent improvements to ntLink have added important features such as overlap detection, gap-filling, and in-code scaffolding iterations. Here, we present three basic protocols demonstrating how to use each of these new features to yield highly contiguous genome assemblies, while still maintaining ntLink's proven computational efficiency. Further, as we illustrate in the alternate protocols, the lightweight minimizer-based mappings that enable ntLink scaffolding can also be utilized for other downstream applications, such as misassembly detection. With its modularity and multiple modes of execution, ntLink has broad benefit to the genomics community, from genome scaffolding and beyond. ntLink is an open-source project and is freely available from https://github.com/bcgsc/ntLink. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: ntLink scaffolding using overlap detection Basic Protocol 2: ntLink scaffolding with gap-filling Basic Protocol 3: Running in-code iterations of ntLink scaffolding Alternate Protocol 1: Generating long-read to contig mappings with ntLink Alternate Protocol 2: Using ntLink mappings for genome assembly correction with Tigmint-long Support Protocol: Installing ntLink.

Molecular Biology of the Cell (MBoC) was conceived nearly 20 years ago as a journal that would be run by and for cell biologists under the auspices of the American Society for Cell Biology (ASCB). The goal was to provide the cell biology community with a journal that would enhance scientific communication among cell biologists by providing authors with rapid, rigorous, constructive, and fair peer review, as well as editorial decisions that were not constrained by journal space or perceived trends. Over the years, MBoC has come to epitomize these values and in so doing has become a pillar of the cell biology community. Today, the values remain the same, but as MBoC heads into its twentieth year we are happy to highlight what we are doing to be more essential and relevant than ever. ASCB ANNUAL MEETING ISSUE AND COVERAGE This issue is MBoC's third annual special issue focused on the ASCB Annual Meeting. It includes a collection of fascinating essays by the recipients of the E. B. Wilson Medal, the Keith Porter Award, the Women in Cell Biology Awards, the E. E. Just Award, and the Early Career Life Scientist Award. Together with invited Perspective and Retrospective essays by other prominent cell biologists, these articles provide insight into research careers, education, mentoring, diversity, science advocacy, and how key discoveries were made. In addition, for the second year in a row, we have invited chairs of the ASCB Annual Meeting Minisymposia to write reviews of their sessions. These will be published early in 2012. A REVAMPED WEBSITE In August, MBoC launched a new and improved website (www.molbiolcell.org). In addition to a more contemporary look, better and more flexible use of screen real estate, and enhanced functionality, the new website includes links to sites with information about careers, education, ASCB events, and cell biology resources. Other new features and functionality will be added in the coming months.

Protocols presented in Current Protocols in Toxicology sometimes include molecular biological, biochemical, and other biological techniques that may not be fully described in this manual. Although it may be reasonable to assume that readers have at least a basic understanding of these techniques, there are times when a full step-by-step description of a procedure is helpful. This appendix lists common molecular biology techniques (some of which are described in this manual) and provides references to specific units in its sister publications Current Protocols in Molecular Biology, Current Protocols in Immunology, and Current Protocols in Cell Biology, which describe the methods in more detail.

BACKGROUND: Acute myeloid leukemia (AML) is an aggressive hematologic cancer with a notoriously bleak prognosis; for non-M3 AML, the overall 5-year survival rate is ∼30%. While 60-70% of newly diagnosed AML patients will achieve complete remission (CR), half of these patients will experience relapse (secondary resistance) by three years from their diagnosis. Moreover, 30-40% of AML patients present with refractory disease (primary resistance) and cannot respond to frontline treatments. Leukemia stem cells (LSCs) are implicated in both primary and secondary resistance, and their eradication is necessary to maintain CR. LSCs have unique transcriptomes and immunophenotypes, thus can be identified relatively accurately using RNA-Seq (e.g., HOXA9 and MEIS1) and flow cytometry (e.g., CD123, CD244, CLL1, c-KIT, and FLT3). However, despite our being able to identify LSCs, agents directed specifically against LSCs do not yet exist. Moreover, being non-selective in terms of the cells targeted, conventional chemotherapy comes with significant toxicity and offers long-term survival to fewer than 1 out of every 3 patients. Thus, there is a need for LSC-specific agents that are as effective as they are tolerable. OBJECTIVE: Considering previous data from our group implicating innate immunity-associated signaling (the TLR-TAK1/TICAM-1 axes) in the survival of LSCs, we sought to characterize TANK-binding kinase 1 (TBK1), a Ser/Thr kinase of the innate immune system, in normal hematopoiesis and MLL-AF9-driven (MLL-AF9+) murine leukemia, to determine if TBK1 may be a feasible drug target for the treatment of AML. METHODS: We generated a tamoxifen-inducible, global Tbk1-knockout (Tbk1NULL) C57BL/6 mouse model. Tbk1fx/fx mice were purchased from Lexicon Pharmaceuticals and crossed with Rosa26-CreERT2+ C57BL/6 mice purchased from The Jackson Laboratory. Isolated HSPCs were transduced with an MSCV retrovirus harboring the MLL-AF9 leukemic oncogene. All procedures were performed in accordance with the NIH's Guide for the Care and Use of Laboratory Animals and an Institutional Animal Care and Use Committee (IACUC)-approved protocol (#2020010) at Loyola University Chicago. RESULTS: In normal murine physiology, we found that Tbk1NULL C57BL/6 mice are viable and grossly normal; the genetic loss of Tbk1 does not significantly impede homeostatic hematopoiesis. However, we discovered that global deletion of Tbk1 leads to an increase in the size of the pool of MPP3-type HSPCs in the bone marrow and the number of neutrophils in peripheral blood. Moreover, we report that while TBK1 does not seem to regulate the engraftment nor reconstitution abilities of transplanted bone marrow cells, TBK1 does negatively regulate monocyte production in an HSPC-intrinsic fashion, as wild-type mice lethally conditioned with busulfan and given Tbk1NULL bone marrow displayed increased monocytes in peripheral blood at endpoint compared to mice transplanted with control marrow. In murine leukemia, we found that the genetic loss of Tbk1 strongly reduces the size of the c-Kit+Flt3+ compartment of MLL-AF9+ HSPCs in vitro and in vivo. We also learned that TBK1 negatively regulates the expression of Csf1r on MLL-AF9+ HSPCs and that some mice given Tbk1NULL MLL-AF9+ HSPCs develop a subcutaneous mass of AML cells (chloroma). DISCUSSION: Murine TBK1 appears to be required by specific LSCs yet dispensable in homeostatic hematopoiesis. While our study requires much further investigation and escalation to better-powered models, we are enthusiastic to present some preliminary data that implicate TBK1 as a drug target in the treatment of MLL-AF9+ AML. This work was supported by the NIH's National Heart, Lung, and Blood Institute (NHLBI; R01 HL133560-01 [Jiwang Zhang] & T35 HL120835 [Austin P. Runde, LUC SSOM's STAR/T35 Program]), National Cancer Institute (NCI; R01 CA223194-01 [Jiwang Zhang]), National.

BackgroundPersonalized therapeutic cancer vaccines aim to target and reprogram the host immune system to achieve cancer eradication in situ. Cancer vaccines deliver two main components: immunostimulants (iS) and tumor antigens to reduce tumor burden with a robust T cell response; however, none have reached broad clinical success due to difficulty in vaccine administration, ex vivo cellular manipulation, low clinical efficacy and broad administrative barriers. While most efforts to date have focused on repeated bolus administrations, biomaterial-based vaccine strategies have led to promising clinical translation.MethodsIn light of these challenges, we have designed a clinically-viable platform-based vaccine strategy, termed the NanoLymph, to provide spatiotemporal elution of immunostimulants and tumor antigens locally to recruit and activate antitumor immunity for cancer eradication. Here, we aim to target the release of granulocyte macrophage colony stimulating factor (GM-CSF) and TLR-7/8 agonist Resiquimod (R848) to promote recruitment and activation of dendritic cells (DCs), a key player in antitumor cytotoxicity.ResultsWe demonstrate the NanoLymph as an structurally stable and biocompatible immunostimulatory niche for durable DC-driven tumor specific T-cell mediated cytotoxicity. Additionally, we demonstrate the NanoLymph’s ability to recruit and activate immune cells of interest, activating antitumor immunity against model antigen. Thus, we have provided the framework necessary to develop a personalized therapeutic cancer vaccine for tumor-specific T-cell mediated responses necessary to generate immunological memory.ConclusionsFuture studies will evaluate immunostimulant and tumor antigen biodistribution in vivo and further apply the NanoLymph in a tumor bearing model to effect antitumor cytotoxicity. Ultimately, we aim to develop a personalized platform applicable for every patient of any cancer type aimed at direct clinical translation.Ethics ApprovalThis study was approved by the Houston Methodist Research Institute (HMRI), according to protocols approved by the Institutional Animal Care and Use Committee (IACUC). HMRI’s Animal Welfare Assurance number is A4555-01. HMRI assures strict compliance with all federal regulations and guidelines involving the use of laboratory animals in biomedical research.

Book Reviews

Scientists are becoming increasingly aware of the need to approach their teaching with the same expectations for evidence that they use in their science. They are coming to recognize that, just as in science research, research on undergraduate science education has a literature as well as standards for practice. I (M.L.L.) am a recent convert to this way of thinking. I taught for 30 years using mostly a lecture style and believed myself quite revolutionary when I used projected images to make cell biological points in class. I now teach using the results of discipline-based education research (DBER) and do my best to use instructional strategies that are demonstrated to improve student learning.

I was invited to write this essay on the occasion of my selection as the recipient of the 2012 Bruce Alberts Award for Excellence in Science Education from the American Society for Cell Biology (ASCB). Receiving this award is an enormous honor. When I read the email announcement for the first time, it was more than a surprise to me, it was unbelievable. I joined ASCB in 1996, when I presented a poster and received a travel award. Since then, I have attended almost every ASCB meeting. I will try to use this essay to share with readers one of the best experiences in my life. Because this is an essay, I take the liberty of mixing some of my thoughts with data in a way that it not usual in scientific writing. I hope that this sacrifice of the format will achieve the goal of conveying what I have learned over the past 20 yr, during which time a group of colleagues and friends created a nexus of knowledge and wisdom. We have worked together to build a network capable of sharing and inspiring science all over the world.