Rapid Deployment of Whole Slide Imaging for Primary Diagnosis in Surgical Pathology at Stanford Medicine: Responding to Challenges of the COVID-19 Pandemic.

CONTEXT.­: Stanford Pathology began stepwise subspecialty implementation of whole slide imaging (WSI) in 2018 soon after the first US Food and Drug Administration approval. In 2020, during the COVID-19 pandemic, the Centers for Medicare & Medicaid Services waived the requirement for pathologists to perform diagnostic tests in Clinical Laboratory Improvement Amendments (CLIA)-licensed facilities. This encouraged rapid implementation of WSI across all surgical pathology subspecialties. OBJECTIVE.­: To present our experience with validation and implementation of WSI at a large academic medical center encompassing a caseload of more than 50 000 cases per year. DESIGN.­: Validation was performed independently for 3 subspecialty services with a diagnostic concordance threshold above 95%. Analysis of user experience, staffing, infrastructure, and information technology was performed after department-wide expansion. RESULTS.­: Diagnostic concordance was achieved in 96% of neuropathology cases, 100% of gynecologic pathology cases, and 98% of immunohistochemistry cases. After full implementation, 8 high-capacity scanners were operational, with whole slide images generated on greater than 2000 slides per weekday, accounting for approximately 80% of histologic slides at Stanford Medicine. Multiple modifications in workflow and information technology were needed to improve performance. Within months of full implementation, most attending pathologists and trainees had adopted WSI for primary diagnosis. CONCLUSIONS.­: WSI across all surgical subspecialities is achievable at scale at an academic medical center; however, adoption required flexibility to adjust workflows and develop tailored solutions. WSI at scale supported the health and safety of medical staff while facilitating high-quality patient care and education during COVID-19 restrictions.

Discovery of a Redox Thiol Switch: Implications for Cellular Energy Metabolism.

The redox-based modifications of cysteine residues in proteins regulate their function in many biological processes. The gas molecule H2S has been shown to persulfidate redox sensitive cysteine residues resulting in an H2S-modified proteome known as the sulfhydrome. Tandem Mass Tags (TMT) multiplexing strategies for large-scale proteomic analyses have become increasingly prevalent in detecting cysteine modifications. Here we developed a TMT-based proteomics approach for selectively trapping and tagging cysteine persulfides in the cellular proteomes. We revealed the natural protein sulfhydrome of two human cell lines, and identified insulin as a novel substrate in pancreatic beta cells. Moreover, we showed that under oxidative stress conditions, increased H2S can target enzymes involved in energy metabolism by switching specific cysteine modifications to persulfides. Specifically, we discovered a Redox Thiol Switch, from protein S-glutathioinylation to S-persulfidation (RTSGS). We propose that the RTSGS from S-glutathioinylation to S-persulfidation is a potential mechanism to fine tune cellular energy metabolism in response to different levels of oxidative stress.

Impact of length of stay on HCAHPS scores following lumbar spine surgery.

OBJECTIVE: Hospital Consumer Assessment of Healthcare Providers and Systems (HCAHPS) surveys, completed by patients following an inpatient stay, are utilized to assess patient satisfaction and quality of the patient experience. HCAHPS results directly impact hospital and provider reimbursements. While recent work has demonstrated that pre- and postoperative factors can affect HCAHPS results following lumbar spine surgery, little is known about how these results are influenced by hospital length of stay (LOS). Here, the authors examined HCAHPS results in patients with LOSs greater or less than expected following lumbar spine surgery to determine whether LOS influences survey scores after these procedures. METHODS: The authors conducted a retrospective review of HCAHPS surveys, patient demographics, and outcomes following lumbar spine surgery at a single institution. A total of 391 patients who had undergone lumbar spine surgery and had completed an HCAHPS survey in the period between 2013 and 2015 were included in this analysis. Patients were divided into those with a hospital LOS equal to or less than the expected (LTE-LOS) and those with a hospital LOS longer than expected (GTE-LOS). Expected LOS was based on the University HealthSystem Consortium benchmarks. Nineteen questions from the HCAHPS survey were examined in relation to patient LOS. The primary outcome measure was a comparison of "top-box" ("9-10" or "always or usually") versus "low-box" ("1-8" and "somewhat or never") scores on the HCAHPS questions. Secondary outcomes of interest were whether the comorbid conditions of cancer, chronic renal failure, diabetes, coronary artery disease, hypertension, stroke, or depression occurred differently with respect to LOS. Statistical analysis was performed using Fisher's exact test for the 2 × 2 contingency tables and the chi-square test for categorical variables. RESULTS: Two hundred fifty-seven patients had an LTE-LOS, whereas 134 patients had a GTE-LOS. The only statistically significant difference in preoperative characteristics between the patient groups was hypertension, which correlated to a shorter LOS. A GTE-LOS was associated with a decreased likelihood of a top-box score for the HCAHPS survey items on doctor listening and pain control. CONCLUSIONS: Here, the authors report a decreased likelihood of top-box responses for some HCAHPS questions following lumbar spine surgery if LOS is prolonged. This study highlights the need to further examine the factors impacting LOS, identify patients at risk for long hospital stays, and improve mechanisms to increase the quality and efficiency of care delivered to this patient population.

Homophilic CD44 Interactions Mediate Tumor Cell Aggregation and Polyclonal Metastasis in Patient-Derived Breast Cancer Models.

Circulating tumor cells (CTC) seed cancer metastases; however, the underlying cellular and molecular mechanisms remain unclear. CTC clusters were less frequently detected but more metastatic than single CTCs of patients with triple-negative breast cancer and representative patient-derived xenograft models. Using intravital multiphoton microscopic imaging, we found that clustered tumor cells in migration and circulation resulted from aggregation of individual tumor cells rather than collective migration and cohesive shedding. Aggregated tumor cells exhibited enriched expression of the breast cancer stem cell marker CD44 and promoted tumorigenesis and polyclonal metastasis. Depletion of CD44 effectively prevented tumor cell aggregation and decreased PAK2 levels. The intercellular CD44-CD44 homophilic interactions directed multicellular aggregation, requiring its N-terminal domain, and initiated CD44-PAK2 interactions for further activation of FAK signaling. Our studies highlight that CD44+ CTC clusters, whose presence is correlated with a poor prognosis of patients with breast cancer, can serve as novel therapeutic targets of polyclonal metastasis. SIGNIFICANCE: CTCs not only serve as important biomarkers for liquid biopsies, but also mediate devastating metastases. CD44 homophilic interactions and subsequent CD44-PAK2 interactions mediate tumor cluster aggregation. This will lead to innovative biomarker applications to predict prognosis, facilitate development of new targeting strategies to block polyclonal metastasis, and improve clinical outcomes.See related commentary by Rodrigues and Vanharanta, p. 22.This article is highlighted in the In This Issue feature, p. 1.

Chemical disruption of the pyroptotic pore-forming protein gasdermin D inhibits inflammatory cell death and sepsis.

Dysregulation of inflammatory cell death is a key driver of many inflammatory diseases. Pyroptosis, a highly inflammatory form of cell death, uses intracellularly generated pores to disrupt electrolyte homeostasis and execute cell death. Gasdermin D, the pore-forming effector protein of pyroptosis, coordinates membrane lysis and the release of highly inflammatory molecules, such as interleukin-1ß, which potentiate the overactivation of the innate immune response. However, to date, there is no pharmacologic mechanism to disrupt pyroptosis. Here, we identify necrosulfonamide as a direct chemical inhibitor of gasdermin D, the pyroptotic pore-forming protein, which binds directly to gasdermin D to inhibit pyroptosis. Pharmacologic inhibition of pyroptotic cell death by necrosulfonamide is efficacious in sepsis models and suggests that gasdermin D inhibitors may be efficacious clinically in inflammatory diseases.

Unique BIR domain sets determine inhibitor of apoptosis protein-driven cell death and NOD2 complex signal specificity.

The mammalian IAPs, X-linked inhibitor of apoptosis protein (XIAP) and cellular inhibitor of apoptosis protein 1 and 2 (cIAP1 and cIAP2), play pivotal roles in innate immune signaling and inflammatory homeostasis, often working in parallel or in conjunction at a signaling complex. IAPs direct both nucleotide-binding oligomerization domain-containing 2 (NOD2) signaling complexes and cell death mechanisms to appropriately regulate inflammation. Although it is known that XIAP is critical for NOD2 signaling and that the loss of cIAP1 and cIAP2 blunts NOD2 activity, it is unclear whether these three highly related proteins can compensate for one another in NOD2 signaling or in mechanisms governing apoptosis or necroptosis. This potential redundancy is critically important, given that genetic loss of XIAP causes both very early onset inflammatory bowel disease and X-linked lymphoproliferative syndrome 2 (XLP-2) and that the overexpression of cIAP1 and cIAP2 is linked to both carcinogenesis and chemotherapeutic resistance. Given the therapeutic interest in IAP inhibition and the potential toxicities associated with disruption of inflammatory homeostasis, we used synthetic biology techniques to examine the functional redundancies of key domains in the IAPs. From this analysis, we defined the features of the IAPs that enable them to function at overlapping signaling complexes but remain independent and functionally exclusive in their roles as E3 ubiquitin ligases in innate immune and inflammatory signaling.

Live-cell visualization of gasdermin D-driven pyroptotic cell death.

Pyroptosis is a form of cell death important in defenses against pathogens that can also result in a potent and sometimes pathological inflammatory response. During pyroptosis, GSDMD (gasdermin D), the pore-forming effector protein, is cleaved, forms oligomers, and inserts into the membranes of the cell, resulting in rapid cell death. However, the potent cell death induction caused by GSDMD has complicated our ability to understand the biology of this protein. Studies aimed at visualizing GSDMD have relied on expression of GSDMD fragments in epithelial cell lines that naturally lack GSDMD expression and also lack the proteases necessary to cleave GSDMD. In this work, we performed mutagenesis and molecular modeling to strategically place tags and fluorescent proteins within GSDMD that support native pyroptosis and facilitate live-cell imaging of pyroptotic cell death. Here, we demonstrate that these fusion proteins are cleaved by caspases-1 and -11 at Asp-276. Mutations that disrupted the predicted p30-p20 autoinhibitory interface resulted in GSDMD aggregation, supporting the oligomerizing activity of these mutations. Furthermore, we show that these novel GSDMD fusions execute inflammasome-dependent pyroptotic cell death in response to multiple stimuli and allow for visualization of the morphological changes associated with pyroptotic cell death in real time. This work therefore provides new tools that not only expand the molecular understanding of pyroptosis but also enable its direct visualization.

Nucleotide-binding oligomerization domain (NOD) signaling defects and cell death susceptibility cannot be uncoupled in X-linked inhibitor of apoptosis (XIAP)-driven inflammatory disease.

The X-linked inhibitor of apoptosis (XIAP) protein has been identified as a key genetic driver of two distinct inflammatory disorders, X-linked lymphoproliferative syndrome 2 (XLP-2) and very-early-onset inflammatory bowel disease (VEO-IBD). Molecularly, the role of XIAP mutations in the pathogenesis of these disorders is unclear. Recent work has consistently shown XIAP to be critical for signaling downstream of the Crohn's disease susceptibility protein nucleotide-binding oligomerization domain-containing 2 (NOD2); however, the reported effects of XLP-2 and VEO-IBD XIAP mutations on cell death have been inconsistent. In this manuscript, we describe a CRISPR-mediated genetic system for cells of the myeloid lineage in which XIAP alleles can be replaced with disease-associated XIAP variants expressed at endogenous levels to simultaneously study inflammation-related cell death and NOD2 signaling. We show that, consistent with previous studies, NOD2 signaling is critically dependent on the BIR2 domain of XIAP. We further used this system to reconcile the aforementioned inconsistent XIAP cell death data to show that XLP-2 and VEO-IBD XIAP mutations that exhibit a loss-of-function NOD2 phenotype also lower the threshold for inflammatory cell death. Last, we identified and studied three novel patient XIAP mutations and used this system to characterize NOD2 and cell death phenotypes driven by XIAP. The results of this work support the role of XIAP in mediating NOD2 signaling while reconciling the role of XLP-2 and VEO-IBD XIAP mutations in inflammatory cell death and provide a set of tools and framework to rapidly test newly discovered XIAP variants.

Cdk5 disruption attenuates tumor PD-L1 expression and promotes antitumor immunity.

Cancers often evade immune surveillance by adopting peripheral tissue- tolerance mechanisms, such as the expression of programmed cell death ligand 1 (PD-L1), the inhibition of which results in potent antitumor immunity. Here, we show that cyclin-dependent kinase 5 (Cdk5), a serine-threonine kinase that is highly active in postmitotic neurons and in many cancers, allows medulloblastoma (MB) to evade immune elimination. Interferon-γ (IFN-γ)-induced PD-L1 up-regulation on MB requires Cdk5, and disruption of Cdk5 expression in a mouse model of MB results in potent CD4(+) T cell-mediated tumor rejection. Loss of Cdk5 results in persistent expression of the PD-L1 transcriptional repressors, the interferon regulatory factors IRF2 and IRF2BP2, which likely leads to reduced PD-L1 expression on tumors. Our finding highlights a central role for Cdk5 in immune checkpoint regulation by tumor cells.

Synthetic Biology Reveals the Uniqueness of the RIP Kinase Domain.

The RIP kinases (RIPKs) play an essential role in inflammatory signaling and inflammatory cell death. However, the function of their kinase activity has been enigmatic, and only recently has kinase domain activity been shown to be crucial for their signal transduction capacity. Despite this uncertainty, the RIPKs have been the subject of intense pharmaceutical development with a number of compounds currently in preclinical testing. In this work, we seek to determine the functional redundancy between the kinase domains of the four major RIPK family members. We find that although RIPK1, RIPK2, and RIPK4 are similar in that they can all activate NF-κB and induce NF-κB essential modulator ubiquitination, only RIPK2 is a dual-specificity kinase. Domain swapping experiments showed that the RIPK4 kinase domain could be converted to a dual-specificity kinase and is essentially indistinct from RIPK2 in biochemical and molecular activity. Surprisingly, however, replacement of RIPK2's kinase domain with RIPK4's did not complement a nucleotide-binding oligomerization domain 2 signaling or gene expression induction defect in RIPK2(-/-) macrophages. These findings suggest that RIPK2's kinase domain is functionally unique compared with other RIPK family members and that pharmacologic targeting of RIPK2 can be separated from the other RIPKs.

An IκB Kinase-Regulated Feedforward Circuit Prolongs Inflammation.

Loss of NF-κB signaling causes immunodeficiency, whereas inhibition of NF-κB can be efficacious in treating chronic inflammatory disease. Inflammatory NF-κB signaling must therefore be tightly regulated, and although many mechanisms to downregulate NF-κB have been elucidated, there have only been limited studies demonstrating positive feedforward regulation of NF-κB signaling. In this work, we use a bioinformatic and proteomic approach to discover that the IKK family of proteins can phosphorylate the E3 ubiquitin ligase ITCH, a critical downregulator of TNF-mediated NF-κB activation. Phosphorylation of ITCH by IKKs leads to impaired ITCH E3 ubiquitin ligase activity and prolongs NF-κB signaling and pro-inflammatory cytokine release. Since genetic loss of ITCH mirrors IKK-induced ITCH phosphorylation, we further show that the ITCH(-/-) mouse's spontaneous lung inflammation and subsequent death can be delayed when TNF signaling is genetically deleted. This work identifies a new positive feedforward regulation of NF-κB activation that drives inflammatory disease.

Pattern transformation with DNA circuits.

Readily programmable chemical networks are important tools as the scope of chemistry expands from individual molecules to larger molecular systems. Although many complex systems are constructed using conventional organic and inorganic chemistry, the programmability of biological molecules such as nucleic acids allows for precise, high-throughput and automated design, as well as simple, rapid and robust implementation. Here we show that systematic and quantitative control over the diffusivity and reactivity of DNA molecules yields highly programmable chemical reaction networks (CRNs) that execute at the macroscale. In particular, we designed and implemented non-enzymatic DNA circuits capable of performing pattern-transformation algorithms such as edge detection. We also showed that it is possible to fine-tune and multiplex such circuits. We believe these strategies will provide programmable platforms on which to prototype CRNs, discover bottom-up construction principles and generate patterns in materials.

Generalized bacterial genome editing using mobile group II introns and Cre-lox.

Efficient bacterial genetic engineering approaches with broad-host applicability are rare. We combine two systems, mobile group II introns ('targetrons') and Cre/lox, which function efficiently in many different organisms, into a versatile platform we call GETR (Genome Editing via Targetrons and Recombinases). The introns deliver lox sites to specific genomic loci, enabling genomic manipulations. Efficiency is enhanced by adding flexibility to the RNA hairpins formed by the lox sites. We use the system for insertions, deletions, inversions, and one-step cut-and-paste operations. We demonstrate insertion of a 12-kb polyketide synthase operon into the lacZ gene of Escherichia coli, multiple simultaneous and sequential deletions of up to 120 kb in E. coli and Staphylococcus aureus, inversions of up to 1.2 Mb in E. coli and Bacillus subtilis, and one-step cut-and-pastes for translocating 120 kb of genomic sequence to a site 1.5 Mb away. We also demonstrate the simultaneous delivery of lox sites into multiple loci in the Shewanella oneidensis genome. No selectable markers need to be placed in the genome, and the efficiency of Cre-mediated manipulations typically approaches 100%.

Human muscle-derived cell populations isolated by differential adhesion rates: phenotype and contribution to skeletal muscle regeneration in Mdx/SCID mice.

Muscle-derived stem cells (MDSCs) isolated from murine skeletal tissue by the preplate method have displayed the capability to commit to the myogenic lineage and regenerate more efficiently than myoblasts in skeletal and cardiac muscle in murine Duchenne Muscular Dystrophy mice (mdx). However, until now, these studies have not been translated to human muscle cells. Here, we describe the isolation, by a preplate technique, of candidate human MDSCs, which exhibit myogenic and regenerative characteristics similar to their murine counterparts. Using the preplate isolation method, we compared cells that adhere faster to the flasks, preplate 2 (PP2), and cells that adhere slower, preplate 6 (PP6). The human PP6 cells express several markers of mesenchymal stem cells and are distinct from human PP2 (a myoblast-like population) based on their expression of CD146 and myogenic markers desmin and CD56. After transplantation to the gastrocnemius muscle of mdx/SCID mice, we observe significantly higher levels of PP6 cells participating in muscle regeneration as compared with the transplantation of PP2 cells. This study supports some previous findings related to mouse preplate cells, and also identifies some differences between mouse and human muscle preplate cells.

Automated live cell imaging systems reveal dynamic cell behavior.

Automated time-lapsed microscopy provides unique research opportunities to visualize cells and subcellular components in experiments with time-dependent parameters. As accessibility to these systems is increasing, we review here their use in cell science with a focus on stem cell research. Although the use of time-lapsed imaging to answer biological questions dates back nearly 150 years, only recently have the use of an environmentally controlled chamber and robotic stage controllers allowed for high-throughput continuous imaging over long periods at the cell and subcellular levels. Numerous automated imaging systems are now available from both companies that specialize in live cell imaging and from major microscope manufacturers. We discuss the key components of robots used for time-lapsed live microscopic imaging, and the unique data that can be obtained from image analysis. We show how automated features enhance experimentation by providing examples of uniquely quantified proliferation and migration live cell imaging data. In addition to providing an efficient system that drastically reduces man-hours and consumes fewer laboratory resources, this technology greatly enhances cell science by providing a unique dataset of temporal changes in cell activity.

High harvest yield, high expansion, and phenotype stability of CD146 mesenchymal stromal cells from whole primitive human umbilical cord tissue.

Human umbilical cord blood is an excellent primitive source of noncontroversial stem cells for treatment of hematologic disorders; meanwhile, new stem cell candidates in the umbilical cord (UC) tissue could provide therapeutic cells for nonhematologic disorders. We show novel in situ characterization to identify and localize a panel of some markers expressed by mesenchymal stromal cells (MSCs; CD44, CD105, CD73, CD90) and CD146 in the UC. We describe enzymatic isolation and purification methods of different UC cell populations that do not require manual separation of the vessels and stroma of the coiled, helical-like UC tissue. Unique quantitation of in situ cell frequency and stromal cell counts upon harvest illustrate the potential to obtain high numerical yields with these methods. UC stromal cells can differentiate to the osteogenic and chondrogenic lineages and, under specific culturing conditions, they exhibit high expandability with unique long-term stability of their phenotype. The remarkable stability of the phenotype represents a novel finding for human MSCs, from any source, and supports the use of these cells as highly accessible stromal cells for both basic studies and potentially therapeutic applications such as allogeneic clinical use for musculoskeletal disorders.