Cultivating Voice and Solidarity in Times of Crisis: Ethnographic Online Journaling as a Pedagogical Tool.

Ethnographic journaling can provide students with powerful opportunities to recognize and value their individual and collective perspectives as both observers and analysts of the world around them, especially in times of crisis. In this Perspectives essay, we share our experiences of using the Pandemic Journaling Project platform as a teaching resource in the first years of the COVID-19 pandemic and beyond. We consider various aspects of online ethnographic journaling, including creative teaching strategies, journaling's therapeutic potential, and student perspectives on the opportunity to document their own experiences as a forward-looking form of "archival activism." We also discuss how journaling can create robust ethnographic learning opportunities while at the same time providing a valuable space for connection and social support, especially when classroom dynamics are constrained by crisis conditions. Ethnographic journaling can help students appreciate what it means for ethnographers to bring their whole selves into their qualitative work in ways that can challenge mainstream misconceptions and contribute concrete forms of data and ethnographic insight. Overall, the essay explores how ethnographic journaling can create meaningful and creative opportunities for curricular innovation; generate durable forms of ethnographic insight; and also bring student experiences into the classroom in ways that can help them cultivate their voice, build a sense of solidarity, and potentially ease student distress.

RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia.

RNA methylation at adenosine N6 (m6A) is one of the most common RNA modifications, impacting RNA stability, transport, and translation. Previous studies uncovered RNA destabilization in amyotrophic lateral sclerosis (ALS) models in association with accumulation of the RNA-binding protein TDP43. Here, we show that TDP43 recognizes m6A RNA and that RNA methylation is critical for both TDP43 binding and autoregulation. We also observed extensive RNA hypermethylation in ALS spinal cord, corresponding to methylated TDP43 substrates. Emphasizing the importance of m6A for TDP43 binding and function, we identified several m6A factors that enhance or suppress TDP43-mediated toxicity via single-cell CRISPR-Cas9 in primary neurons. The most promising modifier-the canonical m6A reader YTHDF2-accumulated within ALS spinal neurons, and its knockdown prolonged the survival of human neurons carrying ALS-associated mutations. Collectively, these data show that m6A modifications modulate RNA binding by TDP43 and that m6A is pivotal for TDP43-related neurodegeneration in ALS.

Specific Direct Small Molecule p300/ß-Catenin Antagonists Maintain Stem Cell Potency.

Despite their high degree of identity and even higher homology, the two Kat3 transcriptional coactivators, CBP and p300, have distinct functions, particularly within the Wnt/ß-catenin signaling cascade. ICG-001, by directly binding to CBP but not p300, inhibits CBP/ß-catenin transcription and has served as an invaluable chemical genomic tool to dissect the Wnt signaling cascade and the divergent roles of these two coactivators. However, to date no direct antagonist of the p300/ß-catenin interaction has been reported. We now report the identification and validation of the first highly specific, direct p300/ß-catenin antagonists, YH249/250 and their ability to maintain pluripotency in ESC.

Wnt signaling orchestration with a small molecule DYRK inhibitor provides long-term xeno-free human pluripotent cell expansion.

An optimal culture system for human pluripotent stem cells should be fully defined and free of animal components. To date, most xeno-free culture systems require human feeder cells and/or highly complicated culture media that contain activators of the fibroblast growth factor (FGF) and transforming growth factor-ß (TGFß) signaling pathways, and none provide for replacement of FGF/TGFß ligands with chemical compounds. The Wnt/ß-catenin signaling pathway plays an important role in mouse embryonic stem cells in leukemia inhibitory factor-independent culture; however, the role of Wnt/ß-catenin signaling in human pluripotent stem cell is still poorly understood and controversial because of the dual role of Wnts in proliferation and differentiation. Building on our previous investigations of small molecules modulating Wnt/ß-catenin signaling in mouse embryonic stem cells, we identified a compound, ID-8, that could support Wnt-induced human embryonic stem cell proliferation and survival without differentiation. Dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) is the target of the small molecule ID-8. Its role in human pluripotent cell renewal was confirmed by DYRK knockdown in human embryonic stem cells. Using Wnt and the DYRK inhibitor ID-8, we have developed a novel and simple chemically defined xeno-free culture system that allows for long-term expansion of human pluripotent stem cells without FGF or TGFß activation. These culture conditions do not include xenobiotic supplements, serum, serum replacement, or albumin. Using this culture system, we have shown that several human pluripotent cell lines maintained pluripotency (>20 passages) and a normal karyotype and still retained the ability to differentiate into derivatives of all three germ layers. This Wnt-dependent culture system should provide a platform for complete replacement of growth factors with chemical compounds.

Profibrotic role of miR-154 in pulmonary fibrosis.

In this study, we explored the regulation and the role of up-regulated microRNAs in idiopathic pulmonary fibrosis (IPF), a progressive interstitial lung disease of unknown origin. We analyzed the expression of microRNAs in IPF lungs and identified 43 significantly up-regulated microRNAs. Twenty-four of the 43 increased microRNAs were localized to the chromosome 14q32 microRNA cluster. We validated the increased expression of miR-154, miR-134, miR-299-5p, miR-410, miR-382, miR-409-3p, miR-487b, miR-31, and miR-127 by quantitative RT-PCR and determined that they were similarly expressed in embryonic lungs. We did not find evidence for differential methylation in this region, but analysis of transcription factor binding sites identified multiple SMAD3-binding elements in the 14q32 microRNA cluster. TGF-ß1 stimulation of normal human lung fibroblasts (NHLF) caused up-regulation of microRNAs on chr14q32 that were also increased in IPF lungs. Chromatin immunoprecipitation confirmed binding of SMAD3 to the putative promoter of miR-154. Mir-154 was increased in IPF fibroblasts, and transfection of NHLF with miR-154 caused significant increases in cell proliferation and migration. The increase in proliferation induced by TGF-ß was not observed when NHLF or IPF fibroblasts were transfected with a mir-154 inhibitor. Transfection with miR-154 caused activation of the WNT pathway in NHLF. ICG-001 and XAV939, inhibitors of the WNT/ß-catenin pathway, reduced the proliferative effect of miR-154. The potential role of miR-154, one of multiple chr14q32 microRNA cluster members up-regulated in IPF and a regulator of fibroblast migration and proliferation, should be further explored in IPF.

Kinetic and structural characterization of caspase-3 and caspase-8 inhibition by a novel class of irreversible inhibitors.

Because of their central role in programmed cell death, the caspases are attractive targets for developing new therapeutics against cancer and autoimmunity, myocardial infarction and ischemic damage, and neurodegenerative diseases. We chose to target caspase-3, an executioner caspase, and caspase-8, an initiator caspase, based on the vast amount of information linking their functions to diseases. Through a structure-based drug design approach, a number of novel beta-strand peptidomimetic compounds were synthesized. Kinetic studies of caspase-3 and caspase-8 inhibition were carried out with these urazole ring-containing irreversible peptidomimetics and a known irreversible caspase inhibitor, Z-VAD-fmk. Using a stopped-flow fluorescence assay, we were able to determine individual kinetic parameters of caspase-3 and caspase-8 inhibition by these inhibitors. Z-VAD-fmk and the peptidomimetic inhibitors inhibit caspase-3 and caspase-8 via a three-step kinetic mechanism. Inhibition of both caspase-3 and caspase-8 by Z-VAD-fmk and of caspase-3 by the peptidomimetic inhibitors proceeds via two rapid equilibrium steps followed by a relatively fast inactivation step. However, caspase-8 inhibition by the peptidomimetics goes through a rapid equilibrium step, a slow-binding reversible step, and an extremely slow inactivation step. The crystal structures of inhibitor complexes of caspases-3 and -8 validate the design of the inhibitors by illustrating in detail how they mimic peptide substrates. One of the caspase-8 structures also shows binding at a secondary, allosteric site, providing a possible route to the development of noncovalent small molecule modulators of caspase activity.

Wnt/beta-catenin/CBP signaling maintains long-term murine embryonic stem cell pluripotency.

Embryonic stem cells (ESCs) represent an important research tool and a potential resource for regenerative medicine. Generally, ESCs are cocultured with a supportive feeder cell layer of murine embryonic fibroblasts, which maintain the ESCs' capacity for self-renewal and block spontaneous differentiation. These cumbersome conditions, as well as the risk of xenobiotic contamination of human ESCs grown on murine embryonic fibroblasts, make it a priority to develop chemically defined methods that can be safely used for the expansion of ESCs. Using a high-throughput, cell-based assay, we identified the small molecule IQ-1 that allows for the Wnt/beta-catenin-driven long-term expansion of mouse ESCs and prevents spontaneous differentiation. We demonstrate that IQ-1, by targeting the PR72/130 subunit of the serine/threonine phosphatase PP2A, prevents beta-catenin from switching coactivator usage from CBP to p300. The increase in beta-catenin/CBP-mediated transcription at the expense of beta-catenin/p300-mediated transcription is critical for the maintenance of murine stem cell pluripotency.

Investigating Wnt signaling: a chemogenomic safari.

Chemical genomics is a powerful method to complement more traditional genetic techniques (i.e. knockout mice, siRNA) for the dissection of complex signaling networks. Wnt signaling in mammals is a complex and crucial regulator of diverse functions. The Wnt-beta-catenin pathway initiates a signaling cascade that is crucial in both normal development and the initiation and progression of cancer. A key step in Wnt activation of target genes is the nuclear translocation of beta-catenin and the formation of a complex between beta-catenin and members of the T-cell factor (TCF) family of transcription factors. Using a forward chemical genomics strategy, we identified ICG-001, a selective inhibitor of a subset of Wnt-beta-catenin-driven gene expression. This chemogenomic tool enables us to dissect this complex signaling network and to better understand the role of Wnt signaling in both normal and pathophysiological settings.

NF-kappaB plays a major role during the systemic and local acute inflammatory response following intestinal reperfusion injury.

1 The nuclear translocation of transcription factors may be a critical factor in the intracellular pathway involved in ischaemia/reperfusion (I/R) injury. Here, we examined whether NF-kappaB and AP-1 participated in the cascade of events leading to TNF-alpha production, neutrophil recruitment, tissue injury and lethality following intestinal I/R. 2 The superior mesenteric artery (SMA) of mice was made ischaemic for 60 min followed by 30 min of reperfusion. The effects of NF-kappaB and AP-1 were studied by the administration of the thioredoxin inhibitor, MOL-294 (methyl 4-hydroxy-4-(8-methyl-1,3-dioxo-2-phenyl-2,3,5,8-tetrahydro-1H-[1,2,4]triazolo[1,2-a]pyridazin-5-yl)but-2-ynoate), and the AP-1 inhibitor, PNRI-299 (N-benzyl-2-(3-cyanophenyl)-1,3,7-trioxo-2,3,7,8-tetrahydro-1H-[1,2,4]triazolo[1,2-a]pyridazine-5-carboxamide). After I/R, there was increase of translocation of NF-kappaB, but not of AP-1, in the intestine and lungs, as assessed by a gel shift assay. 3 Treatment with MOL-294 inhibited the increase in vascular permeability, neutrophil accumulation, hemorrhage and proinflammatory cytokine levels, induced by intestinal I/R injury in the intestine. In the lungs, MOL-294 partially inhibited edema formation, TNF-alpha production, but did not alter neutrophil recruitment. 4 Treatment with MOL-294 inhibited reperfusion-associated lethality, an effect likely to be secondary to the inhibition of systemic TNF-alpha levels. PNRI-299 had no effects on the inflammatory changes or lethality induced by I/R injury. 5 Our results point to an important role for NF-kappaB in triggering endogenous proinflammatory networks during intestinal I/R injury. Inhibition of NF-kappaB prevents tissue injury and lethality, and this was associated with inhibition of TNF-alpha production and decrease in neutrophil recruitment.

Chemogenomics with peptide secondary structure mimetics.

There is increasing evidence that redox regulation of transcription, particularly activator protein-1 (AP-1) and nuclear factor kappa B (NF-kappaB), is important in inflammatory diseases. Human thioredoxin (TRX), a member of the oxidoreductase superfamily, was initially identified, as a factor which augments the production of interleukin-2 receptor alpha (IL-2R alpha) in human T-cell lymphotropic virus type 1 (HTLV-1) infected patient T-cells. Substrates for the redox activity of TRX bind the active site cleft in extended strand structure. The rapid generation of high numbers of peptide secondary structure mimetics through solid-phase synthesis is a key technology for the identification of pharmaceutical leads based on such protein-peptide interactions. In this manuscript, we describe a chemogenomic approach utilizing an extended strand templated library to develop small molecule inhibitors to validate oxidoreductase molecular targets in a murine asthma model.