Immature Lymphocytes Inhibit Rag1 and Rag2 Transcription and V(D)J Recombination in Response to DNA Double-Strand Breaks.

Mammalian cells have evolved a common DNA damage response (DDR) that sustains cellular function, maintains genomic integrity, and suppresses malignant transformation. In pre-B cells, DNA double-strand breaks (DSBs) induced at Igκ loci by the Rag1/Rag2 (RAG) endonuclease engage this DDR to modulate transcription of genes that regulate lymphocyte-specific processes. We previously reported that RAG DSBs induced at one Igκ allele signal through the ataxia telangiectasia mutated (ATM) kinase to feedback-inhibit RAG expression and RAG cleavage of the other Igκ allele. In this article, we show that DSBs induced by ionizing radiation, etoposide, or bleomycin suppress Rag1 and Rag2 mRNA levels in primary pre-B cells, pro-B cells, and pro-T cells, indicating that inhibition of Rag1 and Rag2 expression is a prevalent DSB response among immature lymphocytes. DSBs induced in pre-B cells signal rapid transcriptional repression of Rag1 and Rag2, causing downregulation of both Rag1 and Rag2 mRNA, but only Rag1 protein. This transcriptional inhibition requires the ATM kinase and the NF-κB essential modulator protein, implicating a role for ATM-mediated activation of canonical NF-κB transcription factors. Finally, we demonstrate that DSBs induced in pre-B cells by etoposide or bleomycin inhibit recombination of Igκ loci and a chromosomally integrated substrate. Our data indicate that immature lymphocytes exploit a common DDR signaling pathway to limit DSBs at multiple genomic locations within developmental stages wherein monoallelic Ag receptor locus recombination is enforced. We discuss the implications of our findings for mechanisms that orchestrate the differentiation of monospecific lymphocytes while suppressing oncogenic Ag receptor locus translocations.

R-Spondin-2 Is Upregulated in Idiopathic Pulmonary Fibrosis and Affects Fibroblast Behavior.

Idiopathic pulmonary fibrosis (IPF) is characterized by the expansion of the myofibroblast population, excessive extracellular matrix accumulation, and destruction of the lung parenchyma. The R-spondin family (RSPO) comprises a group of proteins essential for development. Among them, RSPO2 is expressed primarily in the lungs, and its mutations cause severe defects in the respiratory tract. Interestingly, RSPO2 participates in the canonical Wingless/int1 pathway, a critical route in the pathogenesis of IPF. Thus, the aim of this study was to examine the expression and putative role of RSPO2 in this disease. We found that RSPO2 and its receptor leucine-rich G protein-coupled receptor 6 were upregulated in IPF lungs, where they localized primarily in fibroblasts and epithelial cells. Stimulation of IPF and normal lung fibroblasts with recombinant human RSPO2 resulted in the deregulation of numerous genes, although the transcriptional response was essentially distinct. In IPF fibroblasts, RSPO2 stimulation induced the up- or downregulation of several genes involved in the Wingless/int1 pathway (mainly from noncanonical signaling). In both normal and IPF fibroblasts, RSPO2 modifies the expression of genes implicated in several pathways, including the cell cycle and apoptosis. In accordance with gene expression, the stimulation of normal and IPF fibroblasts with RSPO2 significantly reduced cell proliferation and induced cell death. RSPO2 also inhibited collagen production and increased the expression of matrix metalloproteinase 1. Silencing RSPO2 with shRNA induced the opposite effects. Our findings demonstrate, for the first time to our knowledge, that RSPO2 is upregulated in IPF, where it appears to have an antifibrotic role.

Intercellular Communication via the comX-Inducing Peptide (XIP) of Streptococcus mutans.

Gram-positive bacteria utilize exported peptides to coordinate genetic and physiological processes required for biofilm formation, stress responses, and ecological competitiveness. One example is activation of natural genetic competence by ComR and the com X -inducing peptide (XIP) in Streptococcus mutans Although the competence pathway can be activated by the addition of synthetic XIP in defined medium, the hypothesis that XIP is able to function as an intercellular signaling molecule has not been rigorously tested. Coculture model systems were developed that included a "sender" strain that overexpressed the XIP precursor (ComS) and a "responder" strain harboring a green fluorescent protein (GFP) reporter fused to a ComR-activated gene (comX) promoter. The ability of the sender strain to provide a signal to activate GFP expression was monitored at the individual cell and population levels using (i) planktonic culture systems, (ii) cells suspended in an agarose matrix, or (iii) cells growing in biofilms. XIP was shown to be freely diffusible, and XIP signaling between the S. mutans sender and responder strains did not require cell-to-cell contact. The presence of a sucrose-derived exopolysaccharide matrix diminished the efficiency of XIP signaling in biofilms, possibly by affecting the spatial distribution of XIP senders and potential responders. Intercellular signaling was greatly impaired in a strain lacking the primary autolysin, AtlA, and was substantially greater when the sender strain underwent lysis. Collectively, these data provide evidence that S. mutans XIP can indeed function as a peptide signal between cells and highlight the importance of studying signaling with an endogenously produced peptide(s) in populations in various environments and physiologic states.IMPORTANCE The comX-inducing peptide (XIP) of Streptococcus mutans is a key regulatory element in the activation of genetic competence, which allows cells to take up extracellular DNA. XIP has been found in cell culture fluids, and the addition of synthetic XIP to physiologically receptive cells can robustly induce competence gene expression. However, there is a lack of consensus as to whether XIP can function as an intercellular communication signal. Here, we show that XIP indeed signals between cells in S. mutans, but that cell lysis may be a critical factor, as opposed to a dedicated secretion/processing system, in allowing for release of XIP into the environment. The results have important implications in the context of the ecology, virulence, and evolution of a ubiquitous human pathogen and related organisms.

Evaluation of a calcium silicate-based cement as a root reinforcement material for endodontically treated maxillary anterior teeth.

STATEMENT OF PROBLEM: Fractures of endodontically treated teeth have been attributed to weakened tooth structure caused by root canal enlargement and post preparation. PURPOSE: The purpose of this in vitro study was to evaluate the fracture resistance of roots filled with either gutta percha, composite resin (LuxaCore Dual), or calcium silicate-based cement (Biodentine). MATERIAL AND METHODS: One hundred twenty freshly extracted, human, permanent maxillary anterior teeth were sorted by type and assigned to 1 of 4 groups (n=30). The teeth in group NT were not endodontically treated and served as the control. The teeth in groups GP, LC, and, BD were accessed and instrumented to size 40/06. In group GP, the root canals were completely filled with gutta percha. In groups LC and BD, only the apical 5-mm portion of the root canals was filled with gutta percha, and the remaining portion of the root canals was filled with (LuxaCore Dual) in group LC and with (Biodentine) in group BD. Fracture resistance (kN) was assessed at the middle portion of each root, using a 3-point bending test with a universal testing machine exerting a compressive load on a loading pin at a crosshead speed of 0.5 mm/min until fracture occurred. One-way ANOVA was used to compare the mean root fracture resistance among the 4 groups (α=.05). RESULTS: The mean ± SD fracture loads were 2.13 ±0.53 kN for group NT, 1.97 ±0.60 kN for group GP, 2.18 ±0.71 kN for group LC, and 2.22 ±0.54 kN for group BD. No statistically significant differences were found among the 4 groups (P>.05). CONCLUSIONS: The roots of endodontically treated maxillary anterior permanent incisors filled with gutta percha, Biodentine, or LuxaCore Dual had resistance to fracture similar to that of teeth that were not endodontically treated.

PulmonDB: a curated lung disease gene expression database.

Chronic Obstructive Pulmonary Disease (COPD) and Idiopathic Pulmonary Fibrosis (IPF) have contrasting clinical and pathological characteristics and interesting whole-genome transcriptomic profiles. However, data from public repositories are difficult to reprocess and reanalyze. Here, we present PulmonDB, a web-based database (http://pulmondb.liigh.unam.mx/) and R library that facilitates exploration of gene expression profiles for these diseases by integrating transcriptomic data and curated annotation from different sources. We demonstrated the value of this resource by presenting the expression of already well-known genes of COPD and IPF across multiple experiments and the results of two differential expression analyses in which we successfully identified differences and similarities. With this first version of PulmonDB, we create a new hypothesis and compare the two diseases from a transcriptomics perspective.

Clinical outcomes for patients classified by fear-avoidance beliefs and centralization phenomenon.

OBJECTIVES: To (1) determine the prevalence of pain pattern classification subgroups (centralization, noncentralization, and not classified) observed during the initial evaluation of patients experiencing high versus low Fear-Avoidance Beliefs Questionnaire for physical activity (FABQ-PA) scores, (2) examine the association between discharge pain intensity and functional status (FS) outcomes based on FABQ-PA and pain pattern subgroups, and (3) compare minimal clinically important improvement for FS and pain intensity for FABQ-PA and pain pattern classification subgroups. DESIGN: Observational cohort design. SETTING: Suburban hospital-based outpatient rehabilitation clinic. PARTICIPANTS: Consecutive patients with low back syndromes (N=238, mean +/- SD, 59.1+/-17.0y; minimum=20, maximum=91). INTERVENTIONS: Interventions were designed to match patient classification by fear-avoidance level and pain pattern. MAIN OUTCOME MEASURES: Two outcome measures were assessed: patient self-reported FS and pain intensity. FS was assessed by using computerized adaptive testing methods. Maximal pain intensity was assessed by using an 11-point numeric pain scale: 0 (no pain) to 10 (worst imaginable pain). RESULTS: There were no differences (chi(2)(2)=3.7, P=.16) in proportion of patients classified by pain pattern experiencing high or low fear-avoidance beliefs. After controlling for the effect of available risk-adjustment variables, only dual-level classification subgroups, symptom acuity, payer type, and intake FS or pain intensity were associated with discharge FS or pain outcomes. The highest proportion of patients achieving minimal clinically important improvement in pain and FS were reported by the following patient subgroup: centralization and low fear. CONCLUSIONS: Pain pattern and FABQ-PA characteristics impacted rehabilitation outcomes. We recommend that both factors be considered when managing patients with low back pain in an effort to optimize rehabilitation outcomes.

Centralization: prevalence and effect on treatment outcomes using a standardized operational definition and measurement method.

STUDY DESIGN: Retrospective, observational cohort design. OBJECTIVES: Purpose 1 was to determine the association between age, symptom chronicity, and prevalence of centralization in a sample of patients with nonserious cervical or lumbar spinal syndromes referred to a hospital-based outpatient rehabilitation clinic. Purpose 2 was to examine if classifying these patients at intake to centralization or noncentralization predicts functional status, pain intensity, and number of treatment visits at discharge from rehabilitation. Purpose 3 was to compare clinically meaningful changes in functional status and pain intensity between patients subgrouped by centralization and noncentralization. BACKGROUND: Variations in operational definitions and measurements used to identify centralization affect patient classification, contribute to variation in reported prevalence rates, and influence treatment strategy and outcome interpretation. Investigating a standardized operational definition and measurement method for centralization may reduce practice and outcomes variation. METHODS AND MEASURES: Adults (n=418) with cervical or low back syndromes (mean +/- SD age, 58 +/- 17 years; range, 19-91 years; 33% male; 76% lumbar symptoms; 53% chronic symptoms) were assessed. Therapists classified patients using a standardized operational definition and method for centralization during initial evaluation. Prevalence rates were calculated for centralization by age and acuity. Multivariate models were used to assess discharge functional status, pain intensity, and visits while controlling important variables. Percentage of patients subgrouped by centralization and noncentralization achieving minimal clinically important differences (MCID) in functional status and pain intensity was assessed. RESULTS: Overall prevalence rate for centralization was 17%, but increased for patients who were younger and reported acute symptoms regardless of body part. For patients with lumbar syndromes, noncentralization was associated with lower discharge functional status and more pain, but not associated with number of visits compared to patients classified as centralization. For patients with cervical syndromes, noncentralization was associated with more pain but not associated with functional status or number of visits compared to patients classified as centralization. Pain pattern classification affected percentage of patients with lumbar and cervical impairment achieving MCID. CONCLUSION: Results supported the clinical use of a standardized definition of centralization to facilitate patient classification and management and interpretation of outcomes.

YAP1-Mediated Suppression of USP31 Enhances NFκB Activity to Promote Sarcomagenesis.

To date, no consistent oncogenic driver mutations have been identified in most adult soft tissue sarcomas; these tumors are thus generally insensitive to existing targeted therapies. Here we investigated alternate mechanisms underlying sarcomagenesis to identify potential therapeutic interventions. Undifferentiated pleomorphic sarcoma (UPS) is an aggressive tumor frequently found in skeletal muscle where deregulation of the Hippo pathway and aberrant stabilization of its transcriptional effector yes-associated protein 1 (YAP1) increases proliferation and tumorigenesis. However, the downstream mechanisms driving this deregulation are incompletely understood. Using autochthonous mouse models and whole genome analyses, we found that YAP1 was constitutively active in some sarcomas due to epigenetic silencing of its inhibitor angiomotin (AMOT). Epigenetic modulators vorinostat and JQ1 restored AMOT expression and wild-type Hippo pathway signaling, which induced a muscle differentiation program and inhibited sarcomagenesis. YAP1 promoted sarcomagenesis by inhibiting expression of ubiquitin-specific peptidase 31 (USP31), a newly identified upstream negative regulator of NFκB signaling. Combined treatment with epigenetic modulators effectively restored USP31 expression, resulting in decreased NFκB activity. Our findings highlight a key underlying molecular mechanism in UPS and demonstrate the potential impact of an epigenetic approach to sarcoma treatment.Significance: A new link between Hippo pathway signaling, NFκB, and epigenetic reprogramming is highlighted and has the potential for therapeutic intervention in soft tissue sarcomas. Cancer Res; 78(10); 2705-20. ©2018 AACR.

YAP1 enhances NF-κB-dependent and independent effects on clock-mediated unfolded protein responses and autophagy in sarcoma.

Terminal differentiation opposes proliferation in the vast majority of tissue types. As a result, loss of lineage differentiation is a hallmark of aggressive cancers, including soft tissue sarcomas (STS). Consistent with these observations, undifferentiated pleomorphic sarcoma (UPS), an STS subtype devoid of lineage markers, is among the most lethal sarcomas in adults. Though tissue-specific features are lost in these mesenchymal tumors they are most commonly diagnosed in skeletal muscle, and are thought to develop from transformed muscle progenitor cells. We have found that a combination of HDAC (Vorinostat) and BET bromodomain (JQ1) inhibition partially restores differentiation to skeletal muscle UPS cells and tissues, enforcing a myoblast-like identity. Importantly, differentiation is partially contingent upon downregulation of the Hippo pathway transcriptional effector Yes-associated protein 1 (YAP1) and nuclear factor (NF)-κB. Previously, we observed that Vorinostat/JQ1 inactivates YAP1 and restores oscillation of NF-κB in differentiating myoblasts. These effects correlate with reduced tumorigenesis, and enhanced differentiation. However, the mechanisms by which the Hippo/NF-κB axis impact differentiation remained unknown. Here, we report that YAP1 and NF-κB activity suppress circadian clock function, inhibiting differentiation and promoting proliferation. In most tissues, clock activation is antagonized by the unfolded protein response (UPR). However, skeletal muscle differentiation requires both Clock and UPR activity, suggesting the molecular link between them is unique in muscle. In skeletal muscle-derived UPS, we observed that YAP1 suppresses PERK and ATF6-mediated UPR target expression as well as clock genes. These pathways govern metabolic processes, including autophagy, and their disruption shifts metabolism toward cancer cell-associated glycolysis and hyper-proliferation. Treatment with Vorinostat/JQ1 inhibited glycolysis/MTOR signaling, activated the clock, and upregulated the UPR and autophagy via inhibition of YAP1/NF-κB. These findings support the use of epigenetic modulators to treat human UPS. In addition, we identify specific autophagy, UPR, and muscle differentiation-associated genes as potential biomarkers of treatment efficacy and differentiation.

Representaciones sociales de la discapacidad en personas con enfermedades crónicas y estudiantes de terapia física / Social representations of disability in people with chronic diseases and physical therapy students

Resumen: Objetivo: Analizar las representaciones sociales del concepto "discapacidad" por personas con enfermedades crónicas, que acuden a una clínica de rehabilitación y estudiantes de la carrera en terapia física, con la finalidad de conocer la valoración sociocognitiva de sentido común, desde dos perspectivas complementarias. Materiales y métodos: Trabajo con enfoque mixto, fundamentado desde el paradigma cualitativo. Se usó la teoría de las representaciones sociales desde la orientación estructural de Abric. Los participantes se seleccionaron por muestreo propositivo, contando con la colaboración de 134 personas de México. Las técnicas para recolectar la información fueron listados libres y comparación de pares. Se realizaron análisis procesual y estructural. Resultados: La mayoría de los participantes con enfermedades crónicas basan su representación social en vivencias. El campo de la representación se fija en el cuerpo deteriorado y su interrupción en las actividades de la vida cotidiana. Los estudiantes en terapia física basan su representación en conocimientos del modelo médico para la intervención, además de mostrar un pensamiento crítico en torno a la discapacidad. Conclusiones: Los hallazgos pueden orientar a intervenciones basadas en modelos educativos, que hagan al paciente un agente activo del proceso de recuperación de su salud, para incluirse en el entorno social.

Abstract: Objective: To analyze the social representations of the concept of "disability" by people with chronic diseases who attend a rehabilitation clinic and students of the career in physical therapy, in order to know the commonsense socio-cognitive assessment from two complementary perspectives. Materials and methods: A mixed method approach, based on the qualitative paradigm were use. The theory of social representations were used from the structural orientation of Abric. Participants were selected by purposeful sampling, with the collaboration of 134 people from México. Techniques to collect the information were free list and pairs comparison. Processual and structural analyzes were use. Results: Most participants with chronic diseases base their social representation on experiences. The field of representation fixed on the deteriorated body and its interruption in daily life activities. Students in physical therapy base their representation on knowledge of the medical model for intervention, in addition to showing critical thinking regarding disability. Conclusions: The findings can guide interventions based on educational models that make the patient an active agent in the process of recovering their health, to be included in the social environment.

Genomic Alterations of Non-Coding Regions Underlie Human Cancer: Lessons from T-ALL.

It has been appreciated for decades that somatic genomic alterations that change coding sequences of proto-oncogenes, translocate enhancers/promoters near proto-oncogenes, or create fusion oncogenes can drive cancer by inducing oncogenic activities. An explosion of genome-wide technologies over the past decade has fueled discoveries of the roles of three-dimensional chromosome structure and powerful cis-acting elements (super-enhancers) in regulating gene transcription. In recent years, studies of human T cell acute lymphoblastic leukemia (T-ALL) using genome-wide technologies have provided paradigms for how non-coding genomic region alterations can disrupt 3D chromosome architecture or establish super-enhancers to activate oncogenic transcription of proto-oncogenes. These studies raise important issues to consider with the objective of leveraging basic knowledge into new diagnostic and therapeutic opportunities for cancer patients.

Lymphocyte lineage-specific and developmental stage specific mechanisms suppress cyclin D3 expression in response to DNA double strand breaks.

Mammalian cells are thought to protect themselves and their host organisms from DNA double strand breaks (DSBs) through universal mechanisms that restrain cellular proliferation until DNA is repaired. The Cyclin D3 protein drives G1-to-S cell cycle progression and is required for proliferation of immature T and B cells and of mature B cells during a T cell-dependent immune response. We demonstrate that mouse thymocytes and pre-B cells, but not mature B cells, repress Cyclin D3 protein levels in response to DSBs. This response requires the ATM protein kinase that is activated by DSBs. Cyclin D3 protein loss in thymocytes coincides with decreased association of Cyclin D3 mRNA with the HuR RNA binding protein that ATM regulates. HuR inactivation reduces basal Cyclin D3 protein levels without affecting Cyclin D3 mRNA levels, indicating that thymocytes repress Cyclin D3 expression via ATM-dependent inhibition of Cyclin D3 mRNA translation. In contrast, ATM-dependent transcriptional repression of the Cyclin D3 gene represses Cyclin D3 protein levels in pre-B cells. Retrovirus-driven Cyclin D3 expression is resistant to transcriptional repression by DSBs; this prevents pre-B cells from suppressing Cyclin D3 protein levels and from inhibiting DNA synthesis to the normal extent following DSBs. Our data indicate that immature B and T cells use lymphocyte lineage- and developmental stage-specific mechanisms to inhibit Cyclin D3 protein levels and thereby help prevent cellular proliferation in response to DSBs. We discuss the relevance of these cellular context-dependent DSB response mechanisms in restraining proliferation, maintaining genomic integrity, and suppressing malignant transformation of lymphocytes.

Targeting c-MYC in Platinum-Resistant Ovarian Cancer.

The purpose of this study was to investigate the molecular and therapeutic effects of siRNA-mediated c-MYC silencing in cisplatin-resistant ovarian cancer. Statistical analysis of patient's data extracted from The Cancer Genome Atlas (TCGA) portal showed that the disease-free (DFS) and the overall (OS) survival were decreased in ovarian cancer patients with high c-MYC mRNA levels. Furthermore, analysis of a panel of ovarian cancer cell lines showed that c-MYC protein levels were higher in cisplatin-resistant cells when compared with their cisplatin-sensitive counterparts. In vitro cell viability, growth, cell-cycle progression, and apoptosis, as well as in vivo therapeutic effectiveness in murine xenograft models, were also assessed following siRNA-mediated c-MYC silencing in cisplatin-resistant ovarian cancer cells. Significant inhibition of cell growth and viability, cell-cycle arrest, and activation of apoptosis were observed upon siRNA-mediated c-MYC depletion. In addition, single weekly doses of c-MYC-siRNA incorporated into 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG-2000)-based nanoliposomes resulted in significant reduction in tumor growth. These findings identify c-MYC as a potential therapeutic target for ovarian cancers expressing high levels of this oncoprotein.