Middle and High School LGBTQ Students Report What Makes School LGBTQ-Affirming Across Race/Ethnicity and Gender Identity, a Topic Modeling Method.

BACKGROUND: Affirming spaces have been associated with improved mental health outcomes for lesbian, gay, bisexual, transgender, queer, and questioning (LGBTQ) adolescents. METHODS: With data from adolescents currently enrolled in middle or high school across the United States, this study used topic modeling methods to examine students' reports of what they were looking for in LGBTQ-affirming schools and, separately, the association of LGBTQ-affirming schools with suicide risk reduction. RESULTS: Topic models demonstrated consistent themes in how students determined that their school was affirming, such as LGBTQ clubs, teachers requesting pronouns, pride flags, and accepting peers. Students of color uniquely looked for actionable responses in addressing LGBTQ issues. Transgender and nonbinary students required explicit mention of support for transgender issues. Quantitatively, LGBTQ students who reported that their school was LGBTQ-affirming had 20% lower odds of attempting suicide in the past year (adjusted odds ratio = 0.80). CONCLUSIONS: These findings suggest that schools must be safe for all youth and implementing policies that make LGBTQ students feel seen and supported in their identities is a protective factor for mental health. IMPLICATIONS: School policies must ensure that youth have access to supportive people, symbols of support, and LGBTQ clubs and that they are also salient to LGBTQ students of color and transgender and nonbinary students.

Chemical crosslinking and ligation methods for in vivo analysis of RNA structures and interactions.

RNA structures and interactions in living cells drive a variety of biological processes and play critical roles in physiology and disease states. However, studies of RNA structures and interactions have been challenging due to limitations in available technologies. Direct determination of structures in vitro has been only possible to a small number of RNAs with limited sizes and conformations. We recently introduced two chemical crosslink-ligation techniques that enabled studies of transcriptome-wide secondary and tertiary structures and their dynamics. In a dramatically improved version of the psoralen analysis of RNA interactions and structures (PARIS2) method, we detailed the synthesis and use of amotosalen, a highly soluble psoralen analogue, and enhanced enzymology for higher efficiency duplex capture. We also introduced spatial 2'-hydroxyl acylation reversible crosslinking (SHARC) with exonuclease (exo) trimming, a method which utilizes a novel crosslinker class that targets the 2'-OH to capture three-dimensional (3D) structures. Both are powerful orthogonal approaches for solving in vivo RNA structure and interactions, integrating crosslinking, exo trimming, proximity ligation, and high throughput sequencing. In this chapter, we present a detailed protocol for the methods and highlight steps that outperform existing crosslink-ligation approaches.

DDR1 associates with TRPV4 in cell-matrix adhesions to enable calcium-regulated myosin activity and collagen compaction.

Tissue fibrosis manifests as excessive deposition of compacted, highly aligned collagen fibrils, which interfere with organ structure and function. Cells in collagen-rich lesions often exhibit marked overexpression of discoidin domain receptor 1 (DDR1), which is linked to increased collagen compaction through the association of DDR1 with the Ca2+ -dependent nonmuscle myosin IIA (NMIIA). We examined the functional relationship between DDR1 and the transient receptor potential vanilloid type 4 (TRPV4) channel, a Ca2+ -permeable ion channel that is implicated in collagen compaction. Fibroblasts expressing high levels of DDR1 were used to model cells in lesions with collagen compaction. In these cells, the expression of the ß1 integrin was deleted to simplify studies of DDR1 function. Compared with DDR1 wild-type cells, high DDR1 expression was associated with increased Ca2+ influx through TRPV4, enrichment of TRPV4 in collagen adhesions, and enhanced contractile activity mediated by NMIIA. At cell adhesion sites to collagen, DDR1 associated with TRPV4, which enhanced DDR1-mediated collagen alignment and compaction. We conclude that DDR1 regulates Ca2+ influx through the TRPV4 channel to promote critical, DDR1-mediated processes that are important in lesions with collagen compaction and alignment.

Chemical reversible crosslinking enables measurement of RNA 3D distances and alternative conformations in cells.

Three-dimensional (3D) structures dictate the functions of RNA molecules in a wide variety of biological processes. However, direct determination of RNA 3D structures in vivo is difficult due to their large sizes, conformational heterogeneity, and dynamics. Here we present a method, Spatial 2'-Hydroxyl Acylation Reversible Crosslinking (SHARC), which uses chemical crosslinkers of defined lengths to measure distances between nucleotides in cellular RNA. Integrating crosslinking, exonuclease (exo) trimming, proximity ligation, and high throughput sequencing, SHARC enables transcriptome-wide tertiary structure contact maps at high accuracy and precision, revealing heterogeneous RNA structures and interactions. SHARC data provide constraints that improves Rosetta-based RNA 3D structure modeling at near-nanometer resolution. Integrating SHARC-exo with other crosslinking-based methods, we discover compact folding of the 7SK RNA, a critical regulator of transcriptional elongation. These results establish a strategy for measuring RNA 3D distances and alternative conformations in their native cellular context.

Lipoprotein Insulin Resistance Index Reflects Liver Fat Content in Patients With Nonalcoholic Fatty Liver Disease.

The recently developed lipoprotein insulin resistance index (LP-IR) incorporates lipoprotein particle numbers and sizes and is considered to reflect both hepatic and peripheral IR. As tissue IR is a strong component of nonalcoholic fatty liver disease (NAFLD) pathogenesis, we aimed to assess the degree by which LP-IR associates with hepatic fat content. This was a single-center retrospective analysis of patients with NAFLD. LP-IR, the homeostasis model assessment of insulin resistance (HOMA-IR), and adipose tissue IR (Adipo-IR) were measured simultaneously. Liver fat content was estimated by FibroScan controlled attenuated parameter. Associations were assessed using Spearman's correlation and multivariate linear regression. The study included 61 patients. LP-IR was correlated with HOMA-IR (ρ = 0.30; P = 0.02), typically thought to reflect hepatic IR, but not with Adipo-IR (ρ = 0.15; P = 0.25). Liver fat content was significantly associated with Adipo-IR (ρ = 0.48; P < 0.001), LP-IR (ρ = 0.35; P = 0.005), and to a lesser degree with HOMA-IR (ρ = 0.25; P = 0.051). The association of liver fat with LP-IR was limited to patients without diabetes (ρ = 0.60; P < 0.0001), whereas no association was seen in those with diabetes. In a multivariate model, Adipo-IR, LP-IR, and diabetes were independently associated with liver fat and together explained 35% of the variability in liver fat. Conclusion: LP-IR is a reasonable measure of IR in non-diabetic patients with NAFLD and is associated with hepatic fat content. Although adipose tissue is the major contributor to liver fat, the additional contribution of nonadipose tissues can be easily estimated using LP-IR.

Optimized photochemistry enables efficient analysis of dynamic RNA structuromes and interactomes in genetic and infectious diseases.

Direct determination of RNA structures and interactions in living cells is critical for understanding their functions in normal physiology and disease states. Here, we present PARIS2, a dramatically improved method for RNA duplex determination in vivo with >4000-fold higher efficiency than previous methods. PARIS2 captures ribosome binding sites on mRNAs, reporting translation status on a transcriptome scale. Applying PARIS2 to the U8 snoRNA mutated in the neurological disorder LCC, we discover a network of dynamic RNA structures and interactions which are destabilized by patient mutations. We report the first whole genome structure of enterovirus D68, an RNA virus that causes polio-like symptoms, revealing highly dynamic conformations altered by antiviral drugs and different pathogenic strains. We also discover a replication-associated asymmetry on the (+) and (-) strands of the viral genome. This study establishes a powerful technology for efficient interrogation of the RNA structurome and interactome in human diseases.

Vimentin tunes cell migration on collagen by controlling ß1 integrin activation and clustering.

Vimentin is a structural protein that is required for mesenchymal cell migration and directly interacts with actin, ß1 integrin and paxillin. We examined how these interactions enable vimentin to regulate cell migration on collagen. In fibroblasts, depletion of vimentin increased talin-dependent activation of ß1 integrin by more than 2-fold. Loss of vimentin was associated with reduction of ß1 integrin clustering by 50% and inhibition of paxillin recruitment to focal adhesions by more than 60%, which was restored by vimentin expression. This reduction of paxillin was associated with 65% lower Cdc42 activation, a 60% reduction of cell extension formation and a greater than 35% decrease in cell migration on collagen. The activation of PAK1, a downstream effector of Cdc42, was required for vimentin phosphorylation and filament maturation. We propose that vimentin tunes cell migration through collagen by acting as an adaptor protein for focal adhesion proteins, thereby regulating ß1 integrin activation, resulting in well-organized, mature integrin clusters.This article has an associated First Person interview with the first author of the paper.

MRIP Regulates the Myosin IIA Activity and DDR1 Function to Enable Collagen Tractional Remodeling.

DDR1 is a collagen adhesion-mechanoreceptor expressed in fibrotic lesions. DDR1 mediates non-muscle myosin IIA (NMIIA)-dependent collagen remodeling. We discovered that the myosin phosphatase Rho-interacting protein (MRIP), is enriched in DDR1-NMIIA adhesions on collagen. MRIP regulates RhoA- and myosin phosphatase-dependent myosin activity. We hypothesized that MRIP regulates DDR1-NMIIA interactions to enable cell migration and collagen tractional remodeling. After deletion of MRIP in ß1-integrin null cells expressing DDR1, in vitro wound closure, collagen realignment, and contraction were reduced. Cells expressing DDR1 and MRIP formed larger and more abundant DDR1 clusters on collagen than cells cultured on fibronectin or cells expressing DDR1 but null for MRIP or cells expressing a non-activating DDR1 mutant. Deletion of MRIP reduced DDR1 autophosphorylation and blocked myosin light chain-dependent contraction. Deletion of MRIP did not disrupt the association of DDR1 with NMIIA. We conclude that MRIP regulates NMIIA-dependent DDR1 cluster growth and activation. Accordingly, MRIP may provide a novel drug target for dysfunctional DDR1-related collagen tractional remodeling in fibrosis.

Cooperative roles of PAK1 and filamin A in regulation of vimentin assembly and cell extension formation.

The formation of extensions in cell migration requires tightly coordinated reorganization of all three cytoskeletal polymers but the mechanisms by which intermediate filament networks interact with actin to generate extensions are not well-defined. We examined interactions of the actin binding protein filamin A (FLNA) with vimentin in extension formation by fibroblasts. Knockdown (KD) of vimentin in fibroblasts reduced the lengths of cell extensions by 50% (p < 0.001). After cell binding to fibronectin, there was a time-dependent increase of phosphorylation of serine 39, 56 and 72 in vimentin, which was associated with vimentin filament assembly. Of the FLNA-interacting kinases that could phosphorylate vimentin, we focused on PAK1, which we found by reciprocal immunoprecipitation associated with FLNA. Enzyme inhibitor studies and siRNA KD demonstrated that PAK1 was required for vimentin phosphorylation and formation of cell extensions. In sedimentation assays, vimentin was exclusively detected in the insoluble pellet fraction of cells expressing FLNA while in FLNA KD cells there was increased vimentin in the supernatants of FLN KD cells. Compared with wild type, FLNA KD cells showed loss of phosphorylation of serine 56 and 72 in vimentin and reduced numbers and lengths of cell extensions by >4-fold. We suggest that the association of PAK1 with FLNA enables vimentin phosphorylation and filament assembly, which are important in the development and stabilization of cell extensions during cell migration.

A colorimetric assay system for dopamine using microfluidic paper-based analytical devices.

We report the colorimetric detection of dopamine (DA) on microfluidic paper-based analytical devices (µPADs) using an oxidation-reduction method. Here, dopamine reacts with ferric chloride forming reduced Fe2+ that subsequently reacts with phenanthroline to form the red tris(1,10-phenanthroline)iron(II) complex. The devices were fabricated by wax printing and changes in color intensity were recorded using a common cell phone. Subsequent analysis using Photoshop software, yielded a limit of detection (LOD) for DA of 0.37 µmol/L with a linear range of 0.527-4.75 µmol/L and relative standard deviation of 0.11% (inter-day) and 0.15% (intra-day) for n = 15 paper chips. The effects of detection conditions have been investigated and are discussed. Cow serum samples and human blood serum and plasma samples were detected. The work, herein, demonstrates the potential of this method as a low cost and rapid colorimetric technique to detect DA in real samples.

IL1ß and TNFα promote RANKL-dependent adseverin expression and osteoclastogenesis.

Adseverin is an actin-binding protein involved in osteoclastogenesis, but its role in inflammation-induced bone loss is not well-defined. Here, we examined whether IL1ß and TNFα regulate adseverin expression to control osteoclastogenesis in mouse primary monocytes and RAW264.7 cells. Adseverin was colocalized with subcortical actin filaments and was enriched in the fusopods of fusing cells. In precursor cells, adseverin overexpression boosted the formation of RANKL-induced multinucleated cells. Both IL1ß and TNFα enhanced RANKL-dependent TRAcP activity by 1.6-fold and multinucleated cell formation (cells with ≥3 nuclei) by 2.6- and 3.3-fold, respectively. However, IL1ß and TNFα did not enhance osteoclast formation in adseverin-knockdown cells. RANKL-dependent adseverin expression in bone marrow cells was increased by both IL1ß (5.4-fold) and TNFα (3.3-fold). Luciferase assays demonstrated that this expression involved transcriptional regulation of the adseverin promoter. Activation of the promoter was restricted to a 1118 bp sequence containing an NF-κB binding site, upstream of the transcription start site. TNFα also promoted RANKL-induced osteoclast precursor cell migration. We conclude that IL1ß and TNFα enhance RANKL-dependent expression of adseverin, which contributes to fusion processes in osteoclastogenesis.

Thread/paper- and paper-based microfluidic devices for glucose assays employing artificial neural networks.

This paper describes the fabrication of and data collection from two microfluidic devices: a microfluidic thread/paper based analytical device (µTPAD) and 3D microfluidic paper-based analytical device (µPAD). Flowing solutions of glucose oxidase (GOx), horseradish peroxidase (HRP), and potassium iodide (KI), through each device, on contact with glucose, generated a calibration curve for each platform. The resultant yellow-brown color from the reaction indicates oxidation of iodide to iodine. The devices were dried, scanned, and analyzed yielding a correlation between yellow intensity and glucose concentration. A similar procedure, using an unknown concentration of glucose in artificial urine, is conducted and compared to the calibration curve to obtain the unknown value. Studies to quantify glucose in artificial urine showed good correlation between the theoretical and actual concentrations, as percent differences were ≤13.0%. An ANN was trained on the four-channel CMYK color data from 54 µTPAD and 160 µPAD analysis sites and Pearson correlation coefficients of R = 0.96491 and 0.9739, respectively, were obtained. The ANN was able to correctly classify 94.4% (51 of 54 samples) and 91.2% (146 of 160 samples) of the µTPAD and µPAD analysis sites, respectively. The development of this technology combined with ANN should further facilitate the use of these platforms for colorimetric analysis of other analytes.

SEMA3C drives cancer growth by transactivating multiple receptor tyrosine kinases via Plexin B1.

Growth factor receptor tyrosine kinase (RTK) pathway activation is a key mechanism for mediating cancer growth, survival, and treatment resistance. Cognate ligands play crucial roles in autocrine or paracrine stimulation of these RTK pathways. Here, we show SEMA3C drives activation of multiple RTKs including EGFR, ErbB2, and MET in a cognate ligand-independent manner via Plexin B1. SEMA3C expression levels increase in castration-resistant prostate cancer (CRPC), where it functions to promote cancer cell growth and resistance to androgen receptor pathway inhibition. SEMA3C inhibition delays CRPC and enzalutamide-resistant progression. Plexin B1 sema domain-containing:Fc fusion proteins suppress RTK signaling and cell growth and inhibit CRPC progression of LNCaP xenografts post-castration in vivo SEMA3C inhibition represents a novel therapeutic strategy for treatment of advanced prostate cancer.

Semaphorin 3 C drives epithelial-to-mesenchymal transition, invasiveness, and stem-like characteristics in prostate cells.

Prostate cancer (PCa) is among the most commonly-occurring cancers worldwide and a leader in cancer-related deaths. Local non-invasive PCa is highly treatable but limited treatment options exist for those with locally-advanced and metastatic forms of the disease underscoring the need to identify mechanisms mediating PCa progression. The semaphorins are a large grouping of membrane-associated or secreted signalling proteins whose normal roles reside in embryogenesis and neuronal development. In this context, semaphorins help establish chemotactic gradients and direct cell movement. Various semaphorin family members have been found to be up- and down-regulated in a number of cancers. One family member, Semaphorin 3 C (SEMA3C), has been implicated in prostate, breast, ovarian, gastric, lung, and pancreatic cancer as well as glioblastoma. Given SEMA3C's roles in development and its augmented expression in PCa, we hypothesized that SEMA3C promotes epithelial-to-mesenchymal transition (EMT) and stem-like phenotypes in prostate cells. In the present study we show that ectopic expression of SEMA3C in RWPE-1 promotes the upregulation of EMT and stem markers, heightened sphere-formation, and cell plasticity. In addition, we show that SEMA3C promotes migration and invasion in vitro and cell dissemination in vivo.

Filamin A regulates the organization and remodeling of the pericellular collagen matrix.

Extracellular matrix remodeling by cell adhesion-related processes is critical for proliferation and tissue homeostasis, but how adhesions and the cytoskeleton interact to organize the pericellular matrix (PCM) is not understood. We examined the role of the actin-binding protein, filamin A (FLNa), in pericellular collagen remodeling. Compared with wild-type (WT), mice with fibroblast-specific deletion of FLNa exhibited higher density but reduced organization of collagen fibers after increased loading of the periodontal ligament for 2 wk. In cultured fibroblasts, FLNa knockdown (KD) did not affect collagen mRNA, but after 24 h of culture, FLNa WT cells exhibited ∼2-fold higher cell-surface collagen KD cells and 13-fold higher levels of activated ß1 integrins. In FLNa WT cells, there was 3-fold more colocalization of talin with pericellular cleaved collagen than in FLNa KD cells. MMP-9 mRNA and protein expression were >2-fold higher in FLNa KD cells than in WT cells. Cathepsin B, which is necessary for intracellular collagen digestion, was >3-fold higher in FLNa WT cells than in KD cells. FLNa WT cells exhibited 2-fold more collagen phagocytosis than KD cells, which involved the FLNa actin-binding domain. Evidently, FLNa regulates PCM remodeling through its effects on degradation pathways that affect the abundance and organization of collagen.-Mezawa, M., Pinto, V. I., Kazembe, M. P., Lee, W. S., McCulloch, C. A. Filamin A regulates the organization and remodeling of the pericellular collagen matrix.

Particle size influences fibronectin internalization and degradation by fibroblasts.

The application of nanotechnology for drug targeting underlines the importance of controlling the kinetics and cellular sites of delivery for optimal therapeutic outcomes. Here we examined the effect of particle size on internalization and degradation of surface-bound fibronectin by fibroblasts using polystyrene nanoparticles (NPs; 51 nm) and microparticles (MPs; 1 µm). Fibronectin was strongly bound by NPs and MPs as assessed by immuno-dot blot analysis (5.1 ± 0.4 × 10(- 5)pg fibronectin per µm(2) of NP surface; 4.2 ± ± 0.3 × 10(-5)pg fibronectin per µm(2) of MP surface; p>0.2). We estimated that ~193 fibronectin molecules bound to a MP compared with 0.6 fibronectin molecules per NP, indicating that ~40% of nanoparticles were not bound by fibronectin. One hour after incubation, fibronectin-coated NPs and MPs were rapidly internalized by Rat-2 fibroblasts. MPs and NPs were engulfed partly by receptor-mediated endocytosis as indicated by decreased uptake when incubated at 4°C, or by depletion of ATP with sodium azide. Pulse-chase experiments showed minimal exocytosis of NPs and MPs. Internalization of NPs and MPs was inhibited by jasplakinolide, whereas internalization of MPs but not NPs was inhibited by latrunculin B and by integrin-blocking antibodies. Extraction of plasma membrane cholesterol with methyl ß-cyclodextrin inhibited internalization of fibronectin-coated NPs but not MPs. Biotinylated fibronectin internalized by cells was extensively degraded on MPs but not NPs. Particle size affects actin and clathrin-dependent internalization mechanisms leading to fibronectin degradation on MPs but not NPs. Thus either prolonged, controlled release or an immediate delivery of drugs can be achieved by adjusting the particle size along with matrix proteins such as FN.

Delineation of key XRCC4/Ligase IV interfaces for targeted disruption of non-homologous end joining DNA repair.

Efficient DNA repair mechanisms frequently limit the effectiveness of chemotherapeutic agents that act through DNA damaging mechanisms. Consequently, proteins involved in DNA repair have increasingly become attractive targets of high-throughput screening initiatives to identify modulators of these pathways. Disruption of the XRCC4-Ligase IV interaction provides a novel means to efficiently halt repair of mammalian DNA double strand break repair; however; the extreme affinity of these proteins presents a major obstacle for drug discovery. A better understanding of the interaction surfaces is needed to provide a more specific target for inhibitor studies. To clearly define key interface(s) of Ligase IV necessary for interaction with XRCC4, we developed a competitive displacement assay using ESI-MS/MS and determined the minimal inhibitory fragment of the XRCC4-interacting region (XIR) capable of disrupting a complex of XRCC4/XIR. Disruption of a single helix (helix 2) within the helix-loop-helix clamp of Ligase IV was sufficient to displace XIR from a preformed complex. Dose-dependent response curves for the disruption of the complex by either helix 2 or helix-loop-helix fragments revealed that potency of inhibition was greater for the larger helix-loop-helix peptide. Our results suggest a susceptibility to inhibition at the interface of helix 2 and future studies would benefit from targeting this surface of Ligase IV to identify modulators that disrupt its interaction with XRCC4. Furthermore, helix 1 and loop regions of the helix-loop-helix clamp provide secondary target surfaces to identify adjuvant compounds that could be used in combination to more efficiently inhibit XRCC4/Ligase IV complex formation and DNA repair.

Interactions between the discoidin domain receptor 1 and ß1 integrin regulate attachment to collagen.

Collagen degradation by phagocytosis is essential for physiological collagen turnover and connective tissue homeostasis. The rate limiting step of phagocytosis is the binding of specific adhesion receptors, which include the integrins and discoidin domain receptors (DDR), to fibrillar collagen. While previous data suggest that these two receptors interact, the functional nature of these interactions is not defined. In mouse and human fibroblasts we examined the effects of DDR1 knockdown and over-expression on ß1 integrin subunit function. DDR1 expression levels were positively associated with enhanced contraction of floating and attached collagen gels, increased collagen binding and increased collagen remodeling. In DDR1 over-expressing cells compared with control cells, there were increased numbers, area and length of focal adhesions immunostained for talin, paxillin, vinculin and activated ß1 integrin. After treatment with the integrin-cleaving protease jararhagin, in comparison to controls, DDR1 over-expressing cells exhibited increased ß1 integrin cleavage at the cell membrane, indicating that DDR1 over-expression affected the access and susceptibility of cell-surface ß1 integrin to the protease. DDR1 over-expression was associated with increased glycosylation of the ß1 integrin subunit, which when blocked by deoxymannojirimycin, reduced collagen binding. Collectively these data indicate that DDR1 regulates ß1 integrin interactions with fibrillar collagen, which positively impacts the binding step of collagen phagocytosis and collagen remodeling.