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Jacob B Hammond, William W Sheaffer, Chad M Teven, Nabil Wasif, Nitin Mishra, Victor J Davila, William J Casey, Thomas M Polveroni, Leah W Moore, Anthony A Smith Department of Surgery, Mayo Clinic, Phoenix, AZ, USACorrespondence: Anthony A Smith Email smith.anthony@mayo.eduBackground: Formative feedback provides low-stakes opportunities for educational improvement. To enrich our basic science didactics, formative feedback measures were incorporated into our didactics using mobile devices.Materials and Methods: Lecture changes included institutional paid access to a commercial question bank, a 5-item in-class pre-didactic quiz curated from the question bank and taken on the resident’s mobile device, and group discussion of quiz topics. An anonymous survey was sent to participating residents.Results: Overall response rate was 71% among residents. All reported that the new lecture format was a valuable addition to the basic science curriculum (100% Agree/Strongly Agree), and formative assessments provided valuable feedback about the progress of their learning (Strongly Agree = 42%, Agree =58%). All residents reported that in-class use of their mobile device for quizzes was convenient, with majority (84%) preferring it over paper printouts. Residents were more motivated to study before lecture (Strongly Agree = 42%, Agree =42%), with majority also reporting the new format helped identify weaknesses in their knowledgebase (Strongly Agree = 58%, Agree =33%). While majority of residents agreed that quizzes motivated them to study more after lecture, a large portion disagreed (42%). Majority of senior residents reported that the process of composing quizzes prior to lecture enriched their own learning (57%) and helped them find gaps in their knowledge (71%).Conclusion: Incorporating a commercial question bank within didactics gives general surgery residents formative feedback and encourages learning outside the classroom, leading to improved satisfaction with basic science didactics.Keywords: education, surgery, mobile device, question bank, formative feedback

While apoptosis plays a role in B-cell self-tolerance, its significance in preventing autoimmunity remains unclear. Here, we report that dysregulated B cell apoptosis leads to delayed onset autoimmune phenotype in mice. Our longitudinal studies revealed that mice with B cell-specific deletion of pro-apoptotic Bim (BBimfl/fl) have an expanded B cell compartment with a notable increase in transitional, antibody secreting and recently described double negative (DN) B cells. They develop greater hypergammaglobulinemia than mice lacking Bim in all cells and accumulate several autoantibodies characteristic of Systemic Lupus Erythematosus (SLE) and related Sjögren’s Syndrome (SS) including anti-nuclear, anti-Ro/SSA and anti-La/SSB at a level comparable to NODH2h4 autoimmune mouse model. Furthermore, lymphocytes infiltrated the tissues including submandibular glands and formed follicle-like structures populated with B cells, plasma cells and T follicular helper cells indicative of ongoing immune reaction. This autoimmunity was ameliorated upon deletion of Bruton’s tyrosine kinase (Btk) gene, which encodes a key B cell signaling protein. These studies suggest that Bim-mediated apoptosis suppresses and B cell tyrosine kinase signaling promotes B cell-mediated autoimmunity.

Joshua R Niska,1 Sameer R Keole,1 Barbara A Pockaj,2 Michele Y Halyard,1 Samir H Patel,1 Donald W Northfelt,3 Richard J Gray,2 Nabil Wasif,2 Carlos E Vargas,1 William W Wong1 1Department of Radiation Oncology, 2Division of General Surgery, 3Division of Hematology and Medical Oncology, Mayo Clinic Hospital, Phoenix, AZ, USA Background: Recent trials in early-stage breast cancer support hypofractionated whole-breast radiotherapy (WBRT) as part of breast-conserving therapy (BCT). Evidence also suggests that radiotherapy (RT) omission may be reasonable for some patients over 70 years. Among radiation-delivery techniques, intensity-modulated RT (IMRT) is more expensive than 3-dimensional conformal RT (3DCRT). Based on this evidence, in 2013, the American Society for Radiation Oncology (ASTRO) recommended hypofractionated schedules for women aged ≥50 years with early-stage breast cancer and avoiding routine use of IMRT for WBRT. To assess response to level I evidence and adherence to ASTRO recommendations, we evaluated the pattern of RT use for early-stage breast cancer at our National Comprehensive Cancer Network institution from 2006 to 2008 and 2011 to 2013 and compared the results with national trends. Methods: Data from a prospective database were extracted to include patients treated with BCT, aged ≥50 years, with histologic findings of invasive ductal carcinoma, stage T1-T2N0M0, estrogen receptor-positive, and HER2 normal. We retrospectively reviewed the medical records and estimated costs based on 2016 Hospital Outpatient Prospective Payment System (technical fees) and Medicare Physician Fee Schedule (professional fees). Results: Among 55 cases from 2006 to 2008, treatment regimens were 11% hypofractionated, 69% traditional schedule, and 20% RT omission (29% of patients were aged >70 years). Among 83 cases from 2011 to 2013, treatment regimens were 54% hypofractionated, 19% traditional schedule, and 27% RT omission (48% of patients were aged >70 years). 3DCRT was used for all WBRT treatments. Direct medical cost estimates were as follows: 15 fractions 3DCRT, $7,197.87; 15 fractions IMRT, $11,232.33; 25 fractions 3DCRT, $9,731.39; and 25 fractions IMRT, $16,877.45. Conclusion: Despite apparent resistance to shorter radiation schedules in the United States, we demonstrate that rapid practice change in response to level I evidence is feasible. Wider adoption of evidence-based guidelines in early-stage breast cancer may substantially lower health care costs and improve convenience for patients without sacrificing oncologic outcomes. Keywords: breast cancer, CALGB, choosing wisely, hypofractionation, omission, UK START

Splenic transitional B-cells (T1 and T2) are selected to avoid self-reactivity and to safeguard against autoimmunity, then differentiate into mature follicular (FO-I and FO-II) and marginal zone (MZ) subsets. Transcriptomic analysis by RNA-seq of the five B-cell subsets revealed T1 cell signature genes included RAG suggesting a potential for receptor revision. T1 to T2 B-cell differentiation was marked by a switch from Myb to Myc, increased expression of the PI3K adapter DAP10 and MHC class II. FO-II may be an intermediate in FO-I differentiation and may also become MZ B-cells as suggested by principal component analysis (PCA). MZ B-cells possessed the most distinct transcriptome including downregulation of CD45 phosphatase-associated protein (CD45-AP/PTPRC-AP), as well as upregulation of IL-9R and innate molecules TLR3, TLR7 and bactericidal Perforin-2 (MPEG1). Among the endosomal TLRs, stimulation via TLR3 further enhanced Perforin-2 expression exclusively in MZ B-cells. Using gene-deleted and overexpressing transgenic mice we show that IL-9/IL-9R interaction resulted in rapid activation of STAT1, 3 and 5, primarily in MZ B-cells. Importantly, CD45-AP mutant mice had reduced transitional and increased mature MZ and FO B-cells, suggesting that it prevents premature entry of transitional B-cells to the mature B-cell pool or their survival and proliferation. Together, these findings suggest, developmental plasticity among splenic B-cell subsets, potential for receptor revision in peripheral tolerance whereas enhanced metabolism coincides with T2 to mature B-cell differentiation. Further, unique core transcriptional signatures in MZ B-cells may control their innate features.

We describe a previously unappreciated role for Bruton's tyrosine kinase (BTK) in fungal immune surveillance against aspergillosis, an unforeseen complication of BTK inhibitors (BTKi) used for treating B cell lymphoid malignancies. We studied BTK-dependent fungal responses in neutrophils from diverse populations, including healthy donors, patients who were treated with BTKi, and X-linked agammaglobulinemia patients. Upon fungal exposure, BTK was activated in human neutrophils in a TLR2-, Dectin-1-, and Fc[gamma]R-dependent manner, triggering the oxidative burst. BTK inhibition selectively impeded neutrophil-mediated damage to Aspergillus hyphae, primary granule release, and the fungus-induced oxidative burst by abrogating NADPH oxidase subunit [p40.sup.phox] and GTPase RAC2 activation. Moreover, neutrophil-specific Btk deletion in mice enhanced aspergillosis susceptibility by impairing neutrophil function, not recruitment or lifespan. Conversely, GM-CSF partially mitigated these deficits by enhancing [p47.sup.phox] activation. Our findings underline the crucial role of BTK signaling in neutrophils for antifungal immunity and provide a rationale for GM-CSF use to offset these deficits in patients who are susceptible., Introduction Invasive aspergillosis (IA), most often caused by the ubiquitous inhaled mold Aspergillus fumigatus, is an opportunistic fungal infection that exploits numeric or functional neutrophil defects (1-3). IA affects over [...]

Here we show that cells lacking the heme-regulated inhibitor (HRI) are highly resistant to infection by bacterial pathogens. By examining the infection process in wild-type and HRI null cells, we found that HRI is required for pathogens to execute their virulence-associated cellular activities. Specifically, unlike wild-type cells, HRI null cells infected with the gram-negative bacterial pathogen Yersinia are essentially impervious to the cytoskeleton-damaging effects of the Yop virulence factors. This effect is due to reduced functioning of the Yersinia type 3 secretion (T3S) system which injects virulence factors directly into the host cell cytosol. Reduced T3S activity is also observed in HRI null cells infected with the bacterial pathogen Chlamydia which results in a dramatic reduction in its intracellular proliferation. We go on to show that a HRI-mediated process plays a central role in the cellular infection cycle of the Gram-positive pathogen Listeria. For this pathogen, HRI is required for the post-invasion trafficking of the bacterium to the infected host cytosol. Thus by depriving Listeria of its intracellular niche, there is a highly reduced proliferation of Listeria in HRI null cells. We provide evidence that these infection-associated functions of HRI (an eIF2α kinase) are independent of its activity as a regulator of protein synthesis. This is the first report of a host factor whose absence interferes with the function of T3S secretion and cytosolic access by pathogens and makes HRI an excellent target for inhibitors due to its broad virulence-associated activities.

Transphosphorylation by Src family kinases is required for the activation of Bruton's tyrosine kinase (Btk). Differences in the phenotypes of Btk-/- and lyn-/- mice suggest that these kinases may also have independent or opposing functions. B cell development and function were examined in Btk-/-lyn-/- mice to better understand the functional interaction of Btk and Lyn in vivo. The antigen-independent phase of B lymphopoiesis was normal in Btk-/-lyn-/- mice. However, Btk-/-lyn-/- animals had a more severe immunodeficiency than Btk-/- mice. B cell numbers and response to T cell-dependent antigens were reduced. Btk and Lyn therefore play independent or partially redundant roles in the maintenance and function of peripheral B cells. Autoimmunity, hypersensitivity to B cell receptor (BCR) cross-linking, and splenomegaly caused by myeloerythroid hyperplasia were alleviated by Btk deficiency in lyn-/- mice. A transgene expressing Btk at approximately 25% of endogenous levels (Btklo) was crossed onto Btk-/- and Btk-/-lyn-/- backgrounds to demonstrate that Btk is limiting for BCR signaling in the presence but not in the absence of Lyn. These observations indicate that the net outcome of Lyn function in vivo is to inhibit Btk-dependent pathways in B and myeloid cells, and that Btklo mice are a useful sensitized system to identify regulatory components of Btk signaling pathways.

We investigated the role of Bruton's tyrosine kinase (Btk) in FcepsilonRI-dependent activation of mouse mast cells, using xid and btk null mutant mice. Unlike B cell development, mast cell development is apparently normal in these btk mutant mice. However, mast cells derived from these mice exhibited significant abnormalities in FcepsilonRI-dependent function. xid mice primed with anti-dinitrophenyl monoclonal IgE antibody exhibited mildly diminished early-phase and severely blunted late-phase anaphylactic reactions in response to antigen challenge in vivo. Consistent with this finding, cultured mast cells derived from the bone marrow cells of xid or btk null mice exhibited mild impairments in degranulation, and more profound defects in the production of several cytokines, upon FcepsilonRI cross-linking. Moreover, the transcriptional activities of these cytokine genes were severely reduced in FcepsilonRI-stimulated btk mutant mast cells. The specificity of these effects of btk mutations was confirmed by the improvement in the ability of btk mutant mast cells to degranulate and to secrete cytokines after the retroviral transfer of wild-type btk cDNA, but not of vector or kinase-dead btk cDNA. Retroviral transfer of Emt (= Itk/Tsk), Btk's closest relative, also partially improved the ability of btk mutant mast cells to secrete mediators. Taken together, these results demonstrate an important role for Btk in the full expression of FcepsilonRI signal transduction in mast cells.

Space debris is a growing problem for low earth orbit (LEO) and geosynchronous orbit (GEO). The risk of space debris currently affects human activities in Space and is controlled by the collision avoidance alert. However, the risk is growing, which increases future space mission costs to avoid or shield against space debris impact.The project has evolved over four years, culminating in Meng/BEng graduation projects. At the heart of our innovation is utilising the naturally high temperatures in the exosphere and stratosphere, which can soar to 1200 °C. This environment is ideal for initiating a chemical reaction within a pressurised chamber containing a mix of H2-O2 gases, generating heat sufficient to ablate common space debris materials such as titanium, aluminium, and composites. We have crafted an initial satellite design and performed Multiphysics simulations using COMSOL to validate our concept. The project now seeks investment to enhance four critical areas: the satellite's mechanical design to ensure safe operation within a debris field, the development of a dynamic control system for debris collection and satellite navigation, the management of H2 and O2 tank refilling, and the creation of a mechanism for the safe release of ablated materials back into Space.

Blockchain technology has impacted various sectors and is transforming them through its decentralized, immutable, transparent, smart contracts (automatically executing digital agreements) and traceable attributes. Due to the adoption of blockchain technology in versatile applications, millions of transactions take place globally. These transactions are no exception to adversarial attacks which include data tampering, double spending, data corruption, Sybil attacks, eclipse attacks, DDoS attacks, P2P network partitioning, delay attacks, selfish mining, bribery, fake transactions, fake wallets or phishing, false advertising, malicious smart contracts, and initial coin offering scams. These adversarial attacks result in operational, financial, and reputational losses. Although numerous studies have proposed different blockchain anomaly detection mechanisms, challenges persist. These include detecting anomalies in just a single layer instead of multiple layers, targeting a single anomaly instead of multiple, not encountering adversarial machine learning attacks (for example, poisoning, evasion, and model extraction attacks), and inadequate handling of complex transactional data. The proposed AHEAD model solves the above problems by providing the following: (i) data aggregation transformation to detect transactional and user anomalies at the data and network layers of the blockchain, respectively, (ii) a Three-Layer Hierarchical Ensemble Learning Model (HELM) incorporating stratified random sampling to add resilience against adversarial attacks, and (iii) an advanced preprocessing technique with hybrid feature selection to handle complex transactional data. The performance analysis of the proposed AHEAD model shows that it achieves higher anti-adversarial resistance and detects multiple anomalies at the data and network layers. A comparison of the proposed AHEAD model with other state-of-the-art models shows that it achieves 98.85% accuracy against anomaly detection on data and network layers targeting transaction and user anomalies, along with 95.97% accuracy against adversarial machine learning attacks, which surpassed other models.

Mutations in Btk result in the B cell immunodeficiencies X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice. Btk is a critical component of signaling pathways regulating B cell development and function. We used a genetic approach to determine whether Btk is also limiting for these processes. One allele of a murine Btk transgene expressed a dosage of Btk (25% of endogenous levels in splenic B cells) sufficient to restore normal numbers of phenotypically mature conventional B cells in xid mice. 2,4,6-trinitrophenyl-Ficoll response, anti-IgM-induced proliferation, BI cell development, and serum IgM and Ig[G.sub.3] levels remained significantly impaired in these animals. B cells from Btk -/- transgenic mice also responded poorly to anti-IgM, indicating that the xid mutation does not create a dominant negative form of Btk. Response to 2,4,6-trinitrophenyl-Ficoll and B cell receptor cross-linking were increased 3- to 4-fold in xid mice homozygous for the transgene. These results demonstrate that Btk is a limiting component of B cell antigen receptor signaling pathways and suggest that B cell development and response to antigen may require different levels of Btk activity.

Mast cells derived from Bruton's tyrosine kinase (Btk)-defective xid or btk null mice showed greater expansion in culture containing interleukin-3 (IL-3) than those from wild-type (wt) mice. Although the proliferative response to IL-3 was not significantly different between the wt and xid mast cells, xid and btk null mast cells died by apoptosis more slowly than their wt counterparts upon IL-3 deprivation. Consistent with these findings, the apoptosis-linked c-Jun N-terminal kinase/stress-activated protein kinase (JNK) activity was compromised in these btk-mutated cells upon Fc[Epsilon]RI crosslinking or upon stimulation with IL-3 or with stem cell factor. p38 activity was less severely, but significantly, affected by btk mutation, whereas extracellular signal-regulated kinases were not affected by the same mutation. Btk-mediated regulation of apoptosis and JNK activity was confirmed by reconstitution of btk null mutant mast cells with the wt btk cDNA. Furthermore, growth factor withdrawal induced the activation and sustained activity of JNK in wt mast cells, while JNK activity was consistently lower in btk-mutated mast cells. These results support the notion that Btk regulates apoptosis through the JNK activation.