The Exon Junction Complex: A Multitasking Guardian of the Transcriptome.

In a recent issue of Molecular Cell, Boehm et al. (2018), Blazquez et al. (2018), and Gonatopoulos-Pournatzis et al. (2018) uncover novel mechanisms by which the cell regulates splicing of cryptic splice sites and microexons.

Mentoring during Uncertain Times.

Scientific success is mainly supported by mentoring, which often occurs through face-to-face interactions. Changes to the research environment incurred by the Coronavirus 2019 (COVID-19) pandemic have necessitated mentorship adaptations. Here, we describe how mentors can broaden their mentorship to support trainee growth and provide reassurance about trainee development amid uncertain circumstances.

Dysregulation of the chromatin environment leads to differential alternative splicing as a mechanism of disease in a human model of autism spectrum disorder.

Autism spectrum disorder (ASD) affects 1 in 44 children. Chromatin regulatory proteins are overrepresented among genes that contain high risk variants in ASD. Disruption of the chromatin environment leads to widespread dysregulation of gene expression, which is traditionally thought of as a mechanism of disease pathogenesis associated with ASD. Alternatively, alterations in chromatin dynamics could also lead to dysregulation of alternative splicing, which is understudied as a mechanism of ASD pathogenesis. The anticonvulsant valproic acid (VPA) is a well-known environmental risk factor for ASD that acts as a class I histone deacetylase inhibitor. However, the precise molecular mechanisms underlying defects in human neuronal development associated with exposure to VPA are understudied. To dissect how VPA exposure and subsequent chromatin hyperacetylation influence molecular signatures involved in ASD pathogenesis, we conducted RNA sequencing (RNA-seq) in human cortical neurons that were treated with VPA. We observed that differentially expressed genes (DEGs) were enriched for mRNA splicing, mRNA processing, histone modification and metabolism related gene sets. Furthermore, we observed widespread increases in the number and the type of alternative splicing events. Analysis of differential transcript usage (DTU) showed that exposure to VPA induces extensive alterations in transcript isoform usage across neurodevelopmentally important genes. Finally, we find that DEGs and genes that display DTU overlap with known ASD-risk genes. Altogether, these findings suggest that, in addition to differential gene expression, changes in alternative splicing correlated with alterations in the chromatin environment could act as an additional mechanism of disease in ASD.

Treatment strategies for malignant pulmonary nodule: beyond lobectomy. Point-counterpoint.

PURPOSE OF REVIEW: Technological advancement in low-dose computed tomography resulted in an increased incidental discovery of early-stage lung cancer and multifocal ground glass opacity. The demand for parenchyma-preserving treatment strategies is greater now than ever. Pulmonary ablative therapy is a groundbreaking technique to offer local ablative treatment in a lung-sparing manner. It has become a promising technique in lung cancer management with its diverse applicability. In this article, we will review the current development of ablative therapy in lung and look into the future of this innovative technique. RECENT FINDINGS: Current literature suggests that ablative therapy offers comparable local disease control to other local therapies and stereotactic body radiation therapy (SBRT), with a low risk of complications. In particular, bronchoscopic microwave ablation (BMWA) has considerably fewer pleural-based complications due to the avoidance of pleural puncture. BMWA can be considered in the multidisciplinary treatment pathway as it allows re-ablation and allows SBRT after BMWA. SUMMARY: With the benefits which ablative therapy offers and its ability to incorporate into the multidisciplinary management pathway, we foresee ablative therapy, especially BMWA gaining significance in lung cancer treatment. Future directions on developing novel automated navigation platforms and the latest form of ablative energy would further enhance clinical outcomes for our patients.

Home Therapies to Neutralize Button Battery Injury in a Porcine Esophageal Model.

STUDY OBJECTIVE: Button battery ingestion can cause alkaline esophageal injury. There is interest in first-aid household products to neutralize the injury. The objective was to investigate which household products are effective at reducing button battery injury. METHODS: Two cadaveric porcine experiments were performed. Experiment 1 utilized esophageal mucosal segments. A button battery (3VCR2032) was placed onto the mucosa, and substances (saline control, honey, jam, orange juice, yogurt, milk, and cola) were applied every 10 minutes for 6 applications. Tissue pH was measured every 10 minutes, and macroscopic ulceration size was assessed at 120 minutes. Experiment 2 used an intact esophageal model with a battery inserted into the lumen and jam, honey, and saline irrigation as per experiment 1. Tissue pH, macroscopic and histopathology changes were evaluated at 60, 90 and 120 minutes. RESULTS: In experiment 1, only honey and jam had a lower mean tissue pH at 120 minutes (8.0 [standard deviation [SD] 0.9, n=12] and 7.1 [SD 1.7, n=12], respectively) compared to saline solution 11.9 (SD 0.6, n=6, P<.0001). Both honey (0.24 cm2, SD 0.17) and jam (0.37 cm2, SD 0.40) had smaller mean areas of ulceration compared to saline solution (3.90 cm2, SD 1.03, P<.0001). In experiment 2, honey and jam had significantly lower mean tissue pH at all timepoints compared to saline solution. Histologic changes were evident at 60 minutes in the saline group, whereas honey and jam exhibited no or minimal changes until 120 minutes. CONCLUSIONS: Honey and jam were able to neutralize injury caused by a button battery resulting in a smaller area of ulceration. Jam should be further explored as a possible first-aid option as an alternative to honey in suspected button battery ingestion prior to definitive management.

Construction and preliminary validation of a tool to measure the needs of adolescents and young adults (AYA) diagnosed with cancer: the QUestionnaire nEEd Cancer AYAs: QUEEC-AYAs.

CONTEXT: In France, 2300 adolescents and young adults (AYAs, 15-25 years old) are diagnosed with cancer each year. As soon as the disease is diagnosed, a number of physical, psychological and social needs may arise. The aim of this study is to develop a tool to measure unmet needs that will allow the specificities of AYAs to be understood while allowing health care staff to mobilise the necessary actors to resolve them. METHODS: We developed the Questionnaire nEEd Cancer AYAs (QUEEC-AYAs questionnaire), from two existing questionnaires: the Cancer Needs Questionnaire Young People and the Needs Assessment & Service Bridge. A main sample of 103 AYAs then received and completed the questionnaire in order to conduct an exploratory factor analysis. RESULTS: The final structure of the QUEEC-AYAs is composed of 7 dimensions and 48 items: information (8 items), cancer care team (6 items), Physical health (4 items), Emotional health (14 items), Sexual & reproductive health (6 items), Health behaviors & wellness (4 items), Daily life (6 items). The questionnaire has a good acceptability and all domains have a Cronbach's alphas value above 0.80. CONCLUSION: The QUEEC-AYAs is the first measure of the psychosocial needs of AYAs available in French. Its systematic use in health care services should improve the coordination of care required by AYAs during and after treatment. TRIAL REGISTRATION: This study was approved by the ethics committee of the Paoli-Calmettes Institute (IRB # IPC 2021-041, 2021 May 20).

Radiation Principles, Protection, and Reporting for Interventional Pulmonology: A World Association of Bronchology and Interventional Pulmonology White Paper.

The use and availability of diverse advanced X-ray based imaging and guidance systems in the field of interventional pulmonology are rapidly growing. This popularity links inextricably to an increase in ionizing radiation use. Knowing ionizing radiation is hazardous, knowledge and competent use of X-ray imaging and guidance systems are important. The globally implemented As Low As Reasonably Achievable (ALARA) principle demands careful attention to minimize radiation exposure while achieving the precise goals of the intervention and imaging therein. To allow careful and targeted weighing of risk against reward while using X-ray based equipment, proper background knowledge of physics as well as imaging system aspects are needed. This white paper summarizes the principles of ionizing radiation which are crucial to enhance awareness and interpretation of dosimetric quantities. Consecutively, a consensus on standards for reporting radiation exposure in interventional pulmonology procedures is indicated to facilitate comparisons between different systems, approaches and results. Last but not least, it provides a list of practical measures, considerations and tips to optimize procedural imaging as well as reduce radiation dose to patients and staff.

A mouse model of Bardet-Biedl Syndrome has impaired fear memory, which is rescued by lithium treatment.

Primary cilia are microtubule-based organelles present on most cells that regulate many physiological processes, ranging from maintaining energy homeostasis to renal function. However, the role of these structures in the regulation of behavior remains unknown. To study the role of cilia in behavior, we employ mouse models of the human ciliopathy, Bardet-Biedl Syndrome (BBS). Here, we demonstrate that BBS mice have significant impairments in context fear conditioning, a form of associative learning. Moreover, we show that postnatal deletion of BBS gene function, as well as congenital deletion, specifically in the forebrain, impairs context fear conditioning. Analyses indicated that these behavioral impairments are not the result of impaired hippocampal long-term potentiation. However, our results indicate that these behavioral impairments are the result of impaired hippocampal neurogenesis. Two-week treatment with lithium chloride partially restores the proliferation of hippocampal neurons which leads to a rescue of context fear conditioning. Overall, our results identify a novel role of cilia genes in hippocampal neurogenesis and long-term context fear conditioning.

Disruption of dopamine receptor 1 localization to primary cilia impairs signaling in striatal neurons.

A rod-shaped appendage called a primary cilium projects from the soma of most central neurons in the mammalian brain. The importance of cilia within the nervous system is highlighted by the fact that human syndromes linked to primary cilia dysfunction, collectively termed ciliopathies, are associated with numerous neuropathologies, including hyperphagia-induced obesity, neuropsychiatric disorders, and learning and memory deficits. Neuronal cilia are enriched with signaling molecules, including specific G protein-coupled receptors (GPCRs) and their downstream effectors, suggesting they act as sensory organelles that respond to neuromodulators in the extracellular space. We previously showed that GPCR ciliary localization is disrupted in neurons from mouse models of the ciliopathy Bardet-Biedl syndrome (BBS). Based on this finding we hypothesized that mislocalization of ciliary GPCRs may impact receptor signaling and contribute to the BBS phenotypes. Here, we show that disrupting localization of the ciliary GPCR dopamine receptor 1 (D1) in male and female mice, either by loss of a BBS protein or loss of the cilium itself, specifically in D1-expressing neurons, results in obesity. Interestingly, the weight gain is associated with reduced locomotor activity, rather than increased food intake. Moreover, loss of a BBS protein or cilia on D1-expressing neurons leads to a reduction in D1-mediated signaling. Together, these results indicate that cilia impact D1 activity in the nervous system and underscore the importance of neuronal cilia for proper GPCR signaling.SIGNIFICANCE STATEMENT:Most mammalian neurons possess solitary appendages called primary cilia. These rod-shaped structures are enriched with signaling proteins, such as G protein-coupled receptors (GPCRs), suggesting they respond to neuromodulators. This study examines the consequences of disrupting ciliary localization of the GPCR dopamine receptor 1 (D1) in D1-expressing neurons. Remarkably, mice that have either abnormal accumulation of D1 in cilia or loss of D1 ciliary localization become obese. In both cases the obesity is associated with lower locomotor activity rather than overeating. As D1 activation increases locomotor activity, these results are consistent with a reduction in D1 signaling. Indeed, we found that D1-mediated signaling is reduced in brain slices from both mouse models. Thus, cilia impact D1 signaling in the brain.

Mutations in domain IV of elongation factor EF-G confer -1 frameshifting.

A recent crystal structure of a ribosome complex undergoing partial translocation in the absence of elongation factor EF-G showed disruption of codon-anticodon pairing and slippage of the reading frame by -1, directly implicating EF-G in preservation of the translational reading frame. Among mutations identified in a random screen for dominant-lethal mutations of EF-G were a cluster of six that map to the tip of domain IV, which has been shown to contact the codon-anticodon duplex in trapped translocation intermediates. In vitro synthesis of a full-length protein using these mutant EF-Gs revealed dramatically increased -1 frameshifting, providing new evidence for a role for domain IV of EF-G in maintaining the reading frame. These mutations also caused decreased rates of mRNA translocation and rotational movement of the head and body domains of the 30S ribosomal subunit during translocation. Our results are in general agreement with recent findings from Rodnina and coworkers based on in vitro translation of an oligopeptide using EF-Gs containing mutations at two positions in domain IV, who found an inverse correlation between the degree of frameshifting and rates of translocation. Four of our six mutations are substitutions at positions that interact with the translocating tRNA, in each case contacting the RNA backbone of the anticodon loop. We suggest that EF-G helps to preserve the translational reading frame by preventing uncoupled movement of the tRNA through these contacts; a further possibility is that these interactions may stabilize a conformation of the anticodon that favors base-pairing with its codon.

Radical Minimally Invasive Surgery After Immuno-chemotherapy in Initially-unresectable Stage IIIB Non-small cell Lung Cancer.

INTRODUCTION: Use of neoadjuvant immunotherapy agent in advanced stage NSCLC is controversial. Herein, we aim to report on a case series of successful conversion from initial unresectable stage cIIIB NSCLC to radical minimally invasive surgery through immunochemotherapy; with particular attention given to surgical outcomes and survival benefit of surgery. METHODS: Fifty-one patients with initial stage cIIIB NSCLC who received PD-1 agents plus platinum-based chemotherapy between May, 2018 to August, 2020 were retrospectively identified. Surgical and oncological outcomes of enrolled patients were collected. RESULTS: Of 31 patients who underwent subsequent resection, 23 (74.2%) patients underwent lobectomy, 1 (3.2%) underwent pneumonectomy, 5 (16.1%) underwent sleeve lobectomy, and 2 (6.5%) with bilobectomy. The median surgical time was 205 minutes (range, 100-520). The average blood loss was 185 (range: 10-1100) ml. Dense adhesions or fibrosis was noted in 15 cases. The median postoperative hospital stay was 6 (range: 3-13) days. No surgical-related mortality was recorded, only 5 patients (16.1%) experienced any postoperative morbidity (no grade 3 complications). Ten patients (32.3%) had major pathological response, with mediastinal down-staging been observed in 22/31 (71.0%) patients. With a median after up of 15.4 months, thirty-one patients that had surgery had relatively longer median DFS/PFS compared to that of either non-responders or responders that without surgery (27.5 vs. 4.7 vs. 16.7 months, respectively). CONCLUSIONS: Radical surgery after chemoimmunotherapy in initial unresectable stage IIIB NSCLC seems to be safe with low surgical-related mortality and morbidity, and was favorably associated with longer DFS/PFS compared to those without surgery.

Collaborative care for primary care treatment of late-life depression in Singapore: Randomized controlled trial.

BACKGROUND: The effectiveness and portability of the collaborative care model in the primary care treatment of depression has not been demonstrated in many randomized controlled trials in healthcare settings across the world. We determined the effectiveness of collaborative care management of elderly depression in the primary care setting in Singapore. METHOD: Eligible participants with depressive symptoms were randomized to 6-month duration usual care (UC, N = 112) or collaborative care (CC, N = 102). Outcome measures were HDRS-17, GDS, BDI and SF-12 MCS QOL measured at 3, 6, and 12-month, care satisfaction at 6-month, and also measured on 120 participants who refused referral (non-receipt of care, NC). Primary outcome was HDRS-17 measure of depression severity, response and remission at 6-month. RESULTS: HDRS scores in CC group compared to UC group were reduced more at 6-month (1.5 points difference in change from baseline), and also at 3 and 12-month, with similar observations of differences for GDS and BDI. There was significantly greater improvement for both CC and UC groups compared to NC group. The CC group was about 1.5 times more likely to show HDRS treatment response and remission, and more than two times likely to show GDS treatment response and remission than the UC and NC groups, as well as better quality of life improvement (P < .001) and better care satisfaction (P < .001). CONCLUSION: Collaborative care is effective for primary care treatment of older persons with depression and is portable in diverse health care settings.

Electromagnetic Navigation Bronchoscopy Triple Contrast Dye Marking for Lung Nodule Localization.

Small pulmonary lesions can be difficult to localize during video-assisted thoracoscopic surgery. Electromagnetic navigation bronchoscopy (ENB) dye marking of the lesion, particularly when guided by cone beam computed tomography in the hybrid operating room (HOR), is an emerging approach. However, issues with confirmation of dye injection and intraoperative visualization of the colored dye can be unpredictable and challenging. To address these uncertainties, we present our technique of ENB dye marking localization of lung nodule using the triple-contrast dye method in the HOR.

Mutation Profile of Resected EGFR-Mutated Lung Adenocarcinoma by Next-Generation Sequencing.

BACKGROUND: The efficacy of adjuvant targeted therapy for operable lung cancer is still under debate. Comprehensive genetic profiling is needed for detecting co-mutations in resected epidermal growth factor receptor (EGFR)-mutated lung adenocarcinoma (ADC), which may interfere the efficacy of adjuvant tyrosine kinase inhibitor (TKI) treatment. MATERIALS AND METHODS: Mutation profiling of 416 cancer-relevant genes was conducted for 139 resected stage I-IIIa lung ADCs with EGFR mutations using targeted next-generation sequencing. Co-mutation profiles were systematically analyzed. RESULTS: Rare EGFR alterations other than exon 19 deletion and L858R, such as L861Q (∼3%) and G719A (∼2%), were identified at low frequencies. Approximately 10% of patients had mutations in EGFR exon 20 that could confer resistance to first-generation TKIs. Ninety-one percent of patients harbored at least one co-mutation in addition to the major EGFR mutation. TP53 was the top mutated gene and was found more frequently mutated at later stage. Markedly, NF1 mutations were found only in stage II-III ADCs. Conversely, RB1 mutations were more frequent in stage I ADCs, whereas APC mutations were observed exclusively in this group. Thirty-four percent of patients with EGFR TKI-sensitizing mutations had genetic alterations involving EGFR downstream effectors or bypass pathways that could affect the response to EGFR TKIs, such as PIK3CA, BRCA1, and NOTCH1. CONCLUSION: Operable lung ADCs with EGFR TKI-sensitizing mutations are associated with a high proportion of co-mutations. Mutation profiling of these resected tumors could facilitate in determining the applicability and efficacy of adjuvant EGFR TKI therapeutic strategy. IMPLICATIONS FOR PRACTICE: The efficacy of adjuvant epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) therapy for lung cancer harboring EGFR mutation after surgical resection is still under debate. Next-generation sequencing of 416 cancer-relevant genes in 139 resected lung cancers revealed the co-mutational landscape with background EGFR mutation. Notably, the study identified potential EGFR TKI-resistant mutations in 34.71% of patients with a drug-sensitizing EGFR mutation and who were naive in terms of targeted therapy. A comprehensive mutation profiling of these resected tumors could facilitate in determining the applicability and efficacy of adjuvant EGFR TKI therapeutic strategy for these patients.

A Nomogram for Predicting Cancer-Specific Survival of TNM 8th Edition Stage I Non-small-cell Lung Cancer.

BACKGROUND: Models for predicting the survival outcomes of stage I non-small-cell lung cancer (NSCLC) defined by the newly released 8th edition TNM staging system are scarce. This study aimed to develop a nomogram for predicting the cancer-specific survival (CSS) of these patients and identifying individuals with a higher risk for CSS. METHODS: A total of 30,475 NSCLC cases were extracted from the Surveillance, Epidemiology, and End Results (SEER) database. We identified and integrated the risk factors to build a nomogram. The model was subjected to bootstrap internal validation with the SEER database, and external validation with a multicenter cohort of 1133 patients from China. The difference in the impact of adjuvant chemotherapy on model-defined high- and low-risk patients was examined using the National Cancer Database (NCDB). RESULTS: Eight independent prognostic factors were identified and integrated into the model. The calibration curves showed good agreement. The concordance index (C-index) of the nomogram was higher than that of the staging system (IA1, IA2, IA3, and IB) (internal validation set 0.63 vs. 0.56; external validation set 0.66 vs. 0.55; both p < 0.01). Specifically, 21.7% of stage IB patients (7.5% of all stage I) were categorized into the high-risk group (score > 30). There was a significant interaction effect between the adjuvant chemotherapy and risk groups in the NCDB cohort (p = 0.003). CONCLUSIONS: We established a practical nomogram to predict CSS for 8th edition stage I NSCLC. A prospective study is warranted to determine its role in identifying adjuvant chemotherapy candidates.

Assessment of programmed cell death ligand-1 expression by 4 diagnostic assays and its clinicopathological correlation in a large cohort of surgical resected non-small cell lung carcinoma.

Immune checkpoint blockade targeting the PD-1/PD-L1 axis has recently demonstrated efficacy and promise in cancer treatment. Appropriate biomarker selection is therefore essential for improving treatment efficacy. However, the establishment of PD-L1 assay in pathology laboratories is complicated by the presence of multiple testing platforms using different scoring systems. Here we assessed the PD-L1 expression in 713 consecutive non-small cell lung carcinomas by four commercially available PD-L1 immunohistochemical assays, namely, 22C3, 28-8, SP142 and SP263. The analytical performances of the four assays and diagnostic performances across clinically relevant cutoffs were evaluated. The prevalence of PD-L1 (22C3) expression was 21% with a ≥50% cutoff and 56% with a ≥1% cutoff. High PD-L1 expression (using a ≥50% cutoff) was significantly associated with male sex (P = 0.001), ever smoking history (P < 0.001), squamous cell carcinoma (P = 0.001), large cell carcinoma (P < 0.001), lymphoepithelioma-like carcinoma (P = 0.006), sarcomatoid carcinoma (P < 0.001), mutant KRAS (P = 0.005) and wild-type EGFR (P = 0.003). Elevated PD-L1 expression was also significantly associated with shorter survival in patients with adenocarcinoma (log-rank P = 0.026) and remained an independent prognostic factor by multivariable analysis. Among the four assays, 22C3, 28-8 and SP263 were highly concordant for tumor cell scoring. With a cutoff of ≥50% (i.e., the threshold for first-line patient selection), inter-rater agreement was high among the three assays with percentage agreement >97%. In conclusion, three PD-L1 assays showed good analytical performance and a high agreement with each other, but not all cases were correctly classified using the same clinical cutoff. Further studies comparing the predictive value of these assays are required to address the interchangeability of these assays for clinical use.

FOXP3 promotes tumor growth and metastasis by activating Wnt/ß-catenin signaling pathway and EMT in non-small cell lung cancer.

BACKGROUND: The role of cancer cell FOXP3 in tumorigenesis is conflicting. We aimed to study FOXP3 expression and regulation, function and clinical implication in human non-small cell lung cancer (NSCLC). METHODS: One hundred and six patients with histologically-confirmed NSCLC who underwent surgery were recruited for the study. Tumor samples and NSCLC cell lines were used to examine FOXP3 and its related molecules. Various cell functions related to tumorigenesis were performed. In vivo mouse tumor xenograft was used to confirm the in vitro results. RESULTS: NSCLC patients with the high level of FOXP3 had a significant decrease in overall survival and recurrence-free survival. FOXP3 overexpression significantly induced cell proliferation, migration, and invasion, whereas its inhibition impaired its oncogenic function. In vivo studies confirmed that FOXP3 promoted tumor growth and metastasis. The ectopic expression of FOXP3 induced epithelial-mesenchymal transition (EMT) with downregulation of E-cadherin and upregulation of N-cadherin, vimentin, snail, slug, and MMP9. The oncogenic effects by FOXP3 could be attributed to FOX3-mediated activation of Wnt/ß-catenin signaling, as FOXP3 increased luciferase activity of Topflash reporter and upregulated Wnt signaling target genes including c-Myc and Cyclin D1 in NSCLC cells. Co-immunoprecipitation results further indicated that FOXP3 could physically interacted with ß-catenin and TCF4 to enhance the functions of ß-catenin and TCF4, inducing transcription of Wnt target genes to promote cell proliferation, invasion and EMT induction. CONCLUSIONS: FOXP3 can act as a co-activator to facilitate the Wnt-b-catenin signaling pathway, inducing EMT and tumor growth and metastasis in NSCLC.

Comprehensive Proteomic Analysis of Mesenchymal Stem Cell Exosomes Reveals Modulation of Angiogenesis via Nuclear Factor-KappaB Signaling.

Mesenchymal stem cells (MSC) are known to facilitate healing of ischemic tissue related diseases through proangiogenic secretory proteins. Recent studies further show that MSC derived exosomes function as paracrine effectors of angiogenesis, however, the identity of which components of the exosome proteome responsible for this effect remains elusive. To address this we used high-resolution isoelectric focusing coupled liquid chromatography tandem mass spectrometry, an unbiased high throughput proteomics approach to comprehensively characterize the proteinaceous contents of MSCs and MSC derived exosomes. We probed the proteome of MSCs and MSC derived exosomes from cells cultured under expansion conditions and under ischemic tissue simulated conditions to elucidate key angiogenic paracrine effectors present and potentially differentially expressed in these conditions. In total, 6,342 proteins were identified in MSCs and 1,927 proteins in MSC derived exosomes, representing to our knowledge the first time these proteomes have been probed comprehensively. Multilayered analyses identified several putative paracrine effectors of angiogenesis present in MSC exosomes and increased in expression in MSCs exposed to ischemic tissue-simulated conditions; these include platelet derived growth factor, epidermal growth factor, fibroblast growth factor, and most notably nuclear factor-kappaB (NFkB) signaling pathway proteins. NFkB signaling was identified as a key mediator of MSC exosome induced angiogenesis in endothelial cells by functional in vitro validation using a specific inhibitor. Collectively, the results of our proteomic analysis show that MSC derived exosomes contain a robust profile of angiogenic paracrine effectors, which have potential for the treatment of ischemic tissue-related diseases.