A guide for single-particle chromatin tracking in live cell nuclei.

The emergence of labeling strategies and live cell imaging methods enables the imaging of chromatin in living cells at single digit nanometer resolution as well as milliseconds temporal resolution. These technical breakthroughs revolutionize our understanding of chromatin structure, dynamics and functions. Single molecule tracking algorithms are usually preferred to quantify the movement of these intranucleus elements to interpret the spatiotemporal evolution of the chromatin. In this review, we will first summarize the fluorescent labeling strategy of chromatin in live cells which will be followed by a systematic comparison of live cell imaging instrumentation. With the proper microscope, we will discuss the image analysis pipelines to extract the biophysical properties of the chromatin. Finally, we expect to give practical suggestions to broad biologists on how to select methods and link to the model properly according to different investigation purposes.

miR-146a-5p mediates inflammation-induced ß cell mitochondrial dysfunction and apoptosis.

We previously showed that miR-146a-5p is upregulated in pancreatic islets treated with pro-inflammatory cytokines. Others have reported that miR-146a-5p overexpression is associated with ß cell apoptosis and impaired insulin secretion. However, the molecular mechanisms mediating these effects remain elusive. To investigate the role of miR-146a-5p in ß cell function, we developed stable MIN6 cell lines to either overexpress or inhibit the expression of miR-146a-5p. Monoclonal cell populations were treated with pro-inflammatory cytokines (IL-1ß, IFNγ, and TNFα) to model T1D in vitro. We found that overexpression of miR-146a-5p increased cell death under conditions of inflammatory stress, whereas inhibition of miR-146a-5p reversed these effects. Additionally, inhibition of miR-146a-5p increased mitochondrial DNA copy number, respiration rate, and ATP production. Further, RNA sequencing data showed enrichment of pathways related to insulin secretion, apoptosis, and mitochondrial function when the expression levels of miR-146a-5p were altered. Finally, a temporal increase in miR-146a-5p expression levels and a decrease in mitochondria function markers was observed in islets derived from NOD mice. Collectively, these data suggest that miR-146a-5p may promote ß cell dysfunction and death during inflammatory stress by suppressing mitochondrial function.

Pharmacological inhibition of tyrosine protein-kinase 2 reduces islet inflammation and delays type 1 diabetes onset in mice.

Tyrosine protein-kinase 2 (TYK2), a member of the Janus kinase family, mediates inflammatory signaling through multiple cytokines, including interferon-α (IFNα), interleukin (IL)-12, and IL-23. Missense mutations in TYK2 are associated with protection against type 1 diabetes (T1D), and inhibition of TYK2 shows promise in the management of other autoimmune conditions. Here, we evaluated the effects of specific TYK2 inhibitors (TYK2is) in pre-clinical models of T1D. First, human ß cells, cadaveric donor islets, and iPSC-derived islets were treated in vitro with IFNα in combination with a small molecule TYK2i (BMS-986165 or a related molecule BMS-986202). TYK2 inhibition prevented IFNα-induced ß cell HLA class I up-regulation, endoplasmic reticulum stress, and chemokine production. In co-culture studies, pre-treatment of ß cells with a TYK2i prevented IFNα-induced activation of T cells targeting an epitope of insulin. In vivo administration of BMS-986202 in two mouse models of T1D (RIP-LCMV-GP mice and NOD mice) reduced systemic and tissue-localized inflammation, prevented ß cell death, and delayed T1D onset. Transcriptional phenotyping of pancreatic islets, pancreatic lymph nodes (PLN), and spleen during early disease pathogenesis highlighted a role for TYK2 inhibition in modulating signaling pathways associated with inflammation, translational control, stress signaling, secretory function, immunity, and diabetes. Additionally, TYK2i treatment changed the composition of innate and adaptive immune cell populations in the blood and disease target tissues, resulting in an immune phenotype with a diminished capacity for ß cell destruction. Overall, these findings indicate that TYK2i has beneficial effects in both the immune and endocrine compartments in models of T1D, thus supporting a path forward for testing TYK2 inhibitors in human T1D.

Histone Deacetylase Inhibitors Prevent Cytokine-Induced ß Cell Dysfunction Through Restoration of Stromal Interaction Molecule 1 Expression and Activation of Store-Operated Calcium Entry.

Histone deacetylase inhibitors (HDIs) modulate ß cell function in preclinical models of diabetes; however, the mechanisms underlying these beneficial effects have not been determined. In this study, we investigated the impact of the HDI sodium butyrate (NaB) on ß cell function and calcium (Ca2+) signaling using ex vivo and in vitro models of diabetes. Our results show that NaB significantly improved glucose-stimulated insulin secretion in islets from human organ donors with type 2 diabetes and in cytokine-treated INS-1 ß cells. Consistently, NaB partially rescued glucose-stimulated Ca2+ oscillations in mouse islets treated with proinflammatory cytokines. Because the oscillatory phenotype of Ca2+ in the ß cell is governed by changes in endoplasmic reticulum (ER) Ca2+ levels, next we explored the relationship between NaB and store-operated calcium entry (SOCE), a rescue mechanism that acts to refill ER Ca2+ levels through STIM1-mediated gating of plasmalemmal Orai channels. We found that NaB treatment preserved basal ER Ca2+ levels and restored SOCE in IL-1ß-treated INS-1 cells. Furthermore, we linked these changes with the restoration of STIM1 levels in cytokine-treated INS-1 cells and mouse islets, and we found that NaB treatment was sufficient to prevent ß cell death in response to IL-1ß treatment. Mechanistically, NaB counteracted cytokine-mediated reductions in phosphorylation levels of key signaling molecules, including AKT, ERK1/2, glycogen synthase kinase-3α (GSK-3α), and GSK-3ß. Taken together, these data support a model whereby HDI treatment promotes ß cell function and Ca2+ homeostasis under proinflammatory conditions through STIM1-mediated control of SOCE and AKT-mediated inhibition of GSK-3.

ß Cell microRNAs Function as Molecular Hubs of Type 1 Diabetes Pathogenesis and as Biomarkers of Diabetes Risk.

MicroRNAs (miRNAs) are small non-coding RNAs that play a crucial role in modulating gene expression and are enriched in cell-derived extracellular vesicles (EVs). We investigated whether miRNAs from human islets and islet-derived EVs could provide insight into ß cell stress pathways activated during type 1 diabetes (T1D) evolution, therefore serving as potential disease biomarkers. We treated human islets from 10 cadaveric donors with IL-1ß and IFN-γ to model T1D ex vivo. MicroRNAs were isolated from islets and islet-derived EVs, and small RNA sequencing was performed. We found 20 and 14 differentially expressed (DE) miRNAs in cytokine- versus control-treated islets and EVs, respectively. Interestingly, the miRNAs found in EVs were mostly different from those found in islets. Only two miRNAs, miR-155-5p and miR-146a-5p, were upregulated in both islets and EVs, suggesting selective sorting of miRNAs into EVs. We used machine learning algorithms to rank DE EV-associated miRNAs, and developed custom label-free Localized Surface Plasmon Resonance-based biosensors to measure top ranked EVs in human plasma. Results from this analysis revealed that miR-155, miR-146, miR-30c, and miR-802 were upregulated and miR-124-3p was downregulated in plasma-derived EVs from children with recent-onset T1D. In addition, miR-146 and miR-30c were upregulated in plasma-derived EVs of autoantibody positive (AAb+) children compared to matched non-diabetic controls, while miR-124 was downregulated in both T1D and AAb+ groups. Furthermore, single-molecule fluorescence in situ hybridization confirmed increased expression of the most highly upregulated islet miRNA, miR-155, in pancreatic sections from organ donors with AAb+ and T1D.

Protection of ß cells against pro-inflammatory cytokine stress by the GDF15-ERBB2 signaling.

Aim/hypothesis: Growth/differentiation factor 15 (GDF15) is a therapeutic target for a variety of metabolic diseases, including type 1 diabetes (T1D). However, the nausea caused by GDF15 is a challenging point for therapeutic development. In addition, it is unknown why the endogenous GDF15 fails to protect from T1D development. Here, we investigate the GDF15 signaling in pancreatic islets towards opening possibilities for therapeutic targeting in ß cells and to understand why this protection fails to occur naturally. Methods: GDF15 signaling in islets was determined by proximity-ligation assay, untargeted proteomics, pathway analysis, and treatment of cells with specific inhibitors. To determine if GDF15 levels would increase prior to disease onset, plasma levels of GDF15 were measured in a longitudinal prospective study of children during T1D development (n=132 cases vs. n=40 controls) and in children with islet autoimmunity but normoglycemia (n=47 cases vs. n=40 controls) using targeted mass spectrometry. We also investigated the regulation of GDF15 production in islets by fluorescence microscopy and western blot analysis. Results: The proximity-ligation assay identified ERBB2 as the GDF15 receptor in islets, which was confirmed using its specific antagonist, tucatinib. The untargeted proteomics analysis and caspase assay showed that ERBB2 activation by GDF15 reduces ß cell apoptosis by downregulating caspase 8. In plasma, GDF15 levels were higher (p=0.0024) during T1D development compared to controls, but not in islet autoimmunity with normoglycemia. However, in the pancreatic islets GDF15 was depleted via sequestration of its mRNA into stress granules, resulting in translation halting. Conclusions/interpretation: GDF15 protects against T1D via ERBB2-mediated decrease of caspase 8 expression in pancreatic islets. Circulating levels of GDF15 increases pre-T1D onset, which is insufficient to promote protection due to its localized depletion in the islets. These findings open opportunities for targeting GDF15 downstream signaling for pancreatic ß cell protection in T1D and help to explain the lack of natural protection by the endogenous protein.

The Impact of Pro-Inflammatory Cytokines on Alternative Splicing Patterns in Human Islets.

Alternative splicing (AS) within the ß cell has been proposed as one potential pathway that may exacerbate autoimmunity and unveil novel immunogenic epitopes in type 1 diabetes (T1D). We employed a computational strategy to prioritize pathogenic splicing events in human islets treated with IL-1ß + IFN-γ as an ex vivo model of T1D and coupled this analysis with a k-mer based approach to predict RNA binding proteins involved in AS. In total, 969 AS events were identified in cytokine-treated islets, with the majority (44.8%) involving a skipped exon. ExonImpact identified 129 events predicted to impact protein structure. AS occurred with high frequency in MHC Class II-related mRNAs, and targeted qPCR validated reduced inclusion of Exon5 in the MHC Class II gene HLA-DMB. Single molecule RNA FISH confirmed increased HLA-DMB splicing in pancreatic sections from human donors with established T1D and autoantibody positivity. Serine and Arginine Rich Splicing Factor 2 was implicated in 37.2% of potentially pathogenic events, including Exon5 exclusion in HLA-DMB. Together, these data suggest that dynamic control of AS plays a role in the ß cell response to inflammatory signals during T1D evolution.

The Impact of Pro-Inflammatory Cytokines on Alternative Splicing Patterns in Human Islets.

Alternative splicing (AS) within the ß cell has been proposed as one potential pathway that may exacerbate autoimmunity and unveil novel immunogenic epitopes in type 1 diabetes (T1D). We employed a computational strategy to prioritize pathogenic splicing events in human islets treated with IL-1ß + IFN-γ as an ex vivo model of T1D and coupled this analysis with a k-mer based approach to predict RNA binding proteins involved in AS. In total, 969 AS events were identified in cytokine-treated islets, with the majority (44.8%) involving a skipped exon. ExonImpact identified 129 events predicted to impact protein structure. AS occurred with high frequency in MHC Class II-related mRNAs, and targeted qPCR validated reduced inclusion of Exon5 in the MHC Class II gene HLA-DMB. Single molecule RNA FISH confirmed increased HLA-DMB splicing in pancreatic sections from human donors with established T1D and autoantibody positivity. Serine and Arginine Rich Splicing Factor 2 was implicated in 37.2% of potentially pathogenic events, including Exon5 exclusion in HLA-DMB. Together, these data suggest that dynamic control of AS plays a role in the ß cell response to inflammatory signals during T1D evolution.

A multi-institutional phase 2 trial of prostate stereotactic body radiation therapy (SBRT) using continuous real-time evaluation of prostate motion with patient-reported quality of life.

PURPOSE: The use of stereotactic body radiation therapy (SBRT) for prostate cancer has been reported predominantly from single institutional studies, although concerns for broader adoption exist. METHODS AND MATERIALS: From 2011 through 2013, 66 men were accrued to a phase 2 trial at 5 centers. SBRT consisted of 5 fractions of 7.4 Gy to a total dose of 37 Gy using conventional linear accelerators. Electromagnetic transponders were used for motion management. Health-related quality of life (HRQOL) was evaluated via the Expanded Prostate Cancer Index Composite 26 questionnaire. Acute and late toxicities were collected according to Common Terminology Criteria for Adverse Events, version 4.0. Linear mixed modeling was performed to assess changes in HRQOL over time. RESULTS: Median follow-up was 36 months. All men had low- or intermediate-risk disease. There have been 0 biochemical recurrences. No grade 3 urinary or bowel toxicity was reported. Twenty-three percent of patients had acute grade 2 urinary toxicity, with 9% late grade 2 urinary toxicity. Four percent and 5% experienced acute or late grade 2+ bowel toxicity, respectively. Urinary bother and bowel HRQOL transiently decreased during the first 6 to 12 months post-SBRT, and then returned to baseline. In men with good erectile function at baseline, sexual HRQOL declined during the first 6 months and stabilized thereafter. On linear mixed modeling, the strongest predictor of sustained bowel and sexual HRQOL was baseline HRQOL. CONCLUSIONS: In this multi-institutional phase 2 clinical trial using continuous real-time evaluation of prostate motion, prostate SBRT has excellent intermediate-term tumor control with mild and expected treatment-related side effects.

Stereotactic Body Radiation Therapy for Operable Early-Stage Lung Cancer: Findings From the NRG Oncology RTOG 0618 Trial.

Importance: Stereotactic body radiation therapy (SBRT) has become a standard treatment for patients with medically inoperable early-stage lung cancer. However, its effectiveness in patients medically suitable for surgery is unclear. Objective: To evaluate whether noninvasive SBRT delivered on an outpatient basis can safely eradicate lung cancer and cure selected patients with operable lung cancer, obviating the need for surgical resection. Design, Setting, and Participants: Single-arm phase 2 NRG Oncology Radiation Therapy Oncology Group 0618 study enrolled patients from December 2007 to May 2010 with median follow-up of 48.1 months (range, 15.4-73.7 months). The setting was a multicenter North American academic and community practice cancer center consortium. Patients had operable biopsy-proven peripheral T1 to T2, N0, M0 non-small cell tumors no more than 5 cm in diameter, forced expiratory volume in 1 second (FEV1) and diffusing capacity greater than 35% predicted, arterial oxygen tension greater than 60 mm Hg, arterial carbon dioxide tension less than 50 mm Hg, and no severe medical problems. The data analysis was performed in October 2014. Interventions: The SBRT prescription dose was 54 Gy delivered in 3 18-Gy fractions over 1.5 to 2.0 weeks. Main Outcomes and Measures: Primary end point was primary tumor control, with survival, adverse events, and the incidence and outcome of surgical salvage as secondary end points. Results: Of 33 patients accrued, 26 were evaluable (23 T1 and 3 T2 tumors; 15 [58%] male; median age, 72.5 [range, 54-88] years). Median FEV1 and diffusing capacity of the lung for carbon monoxide at enrollment were 72.5% (range, 38%-136%) and 68% (range, 22%-96%) of predicted, respectively. Only 1 patient had a primary tumor recurrence. Involved lobe failure, the other component defining local failure, did not occur in any patient, so the estimated 4-year primary tumor control and local control rate were both 96% (95% CI, 83%-100%). As per protocol guidelines, the single patient with local recurrence underwent salvage lobectomy 1.2 years after SBRT, complicated by a grade 4 cardiac arrhythmia. The 4-year estimates of disease-free and overall survival were 57% (95% CI, 36%-74%) and 56% (95% CI, 35%-73%), respectively. Median overall survival was 55.2 months (95% CI, 37.7 months to not reached). Protocol-specified treatment-related grade 3, 4, and 5 adverse events were reported in 2 (8%; 95% CI, 0.1%-25%), 0, and 0 patients, respectively. Conclusions and Relevance: As given, SBRT appears to be associated with a high rate of primary tumor control, low treatment-related morbidity, and infrequent need for surgical salvage in patients with operable early-stage lung cancer. Trial Registration: ClinicalTrials.gov Identifier: NCT00551369.