The Peptidoglycan Recognition Protein 1 confers immune evasive properties on pancreatic cancer stem cells.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) has limited therapeutic options, particularly with immune checkpoint inhibitors. Highly chemoresistant 'stem-like' cells, known as cancer stem cells (CSCs), are implicated in PDAC aggressiveness. Thus, comprehending how this subset of cells evades the immune system is crucial for advancing novel therapies. DESIGN: We used the KPC mouse model (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre) and primary tumour cell lines to investigate putative CSC populations. Transcriptomic analyses were conducted to pinpoint new genes involved in immune evasion. Overexpressing and knockout cell lines were established with lentiviral vectors. Subsequent in vitro coculture assays, in vivo mouse and zebrafish tumorigenesis studies, and in silico database approaches were performed. RESULTS: Using the KPC mouse model, we functionally confirmed a population of cells marked by EpCAM, Sca-1 and CD133 as authentic CSCs and investigated their transcriptional profile. Immune evasion signatures/genes, notably the gene peptidoglycan recognition protein 1 (PGLYRP1), were significantly overexpressed in these CSCs. Modulating PGLYRP1 impacted CSC immune evasion, affecting their resistance to macrophage-mediated and T-cell-mediated killing and their tumourigenesis in immunocompetent mice. Mechanistically, tumour necrosis factor alpha (TNFα)-regulated PGLYRP1 expression interferes with the immune tumour microenvironment (TME) landscape, promoting myeloid cell-derived immunosuppression and activated T-cell death. Importantly, these findings were not only replicated in human models, but clinically, secreted PGLYRP1 levels were significantly elevated in patients with PDAC. CONCLUSIONS: This study establishes PGLYRP1 as a novel CSC-associated marker crucial for immune evasion, particularly against macrophage phagocytosis and T-cell killing, presenting it as a promising target for PDAC immunotherapy.

Targeting cancer stem cell OXPHOS with tailored ruthenium complexes as a new anti-cancer strategy.

BACKGROUND: Previous studies by our group have shown that oxidative phosphorylation (OXPHOS) is the main pathway by which pancreatic cancer stem cells (CSCs) meet their energetic requirements; therefore, OXPHOS represents an Achille's heel of these highly tumorigenic cells. Unfortunately, therapies that target OXPHOS in CSCs are lacking. METHODS: The safety and anti-CSC activity of a ruthenium complex featuring bipyridine and terpyridine ligands and one coordination labile position (Ru1) were evaluated across primary pancreatic cancer cultures and in vivo, using 8 patient-derived xenografts (PDXs). RNAseq analysis followed by mitochondria-specific molecular assays were used to determine the mechanism of action. RESULTS: We show that Ru1 is capable of inhibiting CSC OXPHOS function in vitro, and more importantly, it presents excellent anti-cancer activity, with low toxicity, across a large panel of human pancreatic PDXs, as well as in colorectal cancer and osteosarcoma PDXs. Mechanistic studies suggest that this activity stems from Ru1 binding to the D-loop region of the mitochondrial DNA of CSCs, inhibiting OXPHOS complex-associated transcription, leading to reduced mitochondrial oxygen consumption, membrane potential, and ATP production, all of which are necessary for CSCs, which heavily depend on mitochondrial respiration. CONCLUSIONS: Overall, the coordination complex Ru1 represents not only an exciting new anti-cancer agent, but also a molecular tool to dissect the role of OXPHOS in CSCs. Results indicating that the compound is safe, non-toxic and highly effective in vivo are extremely exciting, and have allowed us to uncover unprecedented mechanistic possibilities to fight different cancer types based on targeting CSC OXPHOS.

Social Isolation, Healthy Habits, Inequality and Mental Health in the United States.

The objective of this work is to deepen the analysis of the socioeconomic determinants of mental health, paying special attention to the impact of inequality, not only in income distribution but also in gender, racial, health and education inequality, social isolation, including new variables to measure loneliness, and healthy habits, on the mental health status. For this purpose, a cross-sectional model for a sample of 2735 counties in the United States is estimated using Ordinary Least Squares in its robust version to solve the detected heteroscedasticity problems. The results obtained show that inequality, social isolation and certain lifestyles, such as smoking or insomnia, are detrimental to mental health, while sexual activity prevents mental distress. On the other hand, poor counties suffer more cases of suicide, with food insecurity being the main problem for mental health. Finally, we found detrimental effects of pollution on mental health.

Association between the level of indifference to dental treatment and habits of oral hygiene in adults / Asociación entre el nivel de indiferencia al tratamiento dental y los hábitos de higiene bucal

Introduction: Inadequate oral hygiene habits such as lack of tooth brushing, use of irrigators, fluoridated toothpastes, and dental floss, as well as the importance of worrying about going to the dentist in the event of any problem in the oral cavity and the continuity of treatment are factors that could increase the risk in patients. Objetive: To determine the association between the level of indifference to dental treatment and the oral hygiene habits of those surveyed adults. Material and Methods: An observational, analytical and cross-sectional study. The level of indifference to dental treatment was evaluated using a translated virtual questionnaire and subsequently internally validated (Cronbach's alpha: 0.91). There were a total of 249 participants, 150 males and 99 females, with a mean age of 30.69 years. For the association of the qualitative variables and the report of the crude and adjusted odds' ratio (OR), a logistic regression was used. We worked with a level of statistical significance of p<0.05 and a confidence interval of 95%. Results: A high level of indifference to dental treatment was obtained in 57.83% of the respondents. Likewise, a statistically significant association was found between indifference to dental treatment and education (p = 0.012). Regarding oral hygiene habits, a statistically significant association was found with flossing (OR = 2.22; 95% CI: 1.10-4.46) (p = 0.025) and brushing before sleeping (OR = 5.26; 95%: 2.26-12-22) (p<0.001). Conclusion: There is a statistically significant association between the level of indifference to dental treatment with oral hygiene habits, flossing and brushing before sleeping. It is advisable to carry out activities in the communities to promote oral health care to reduce levels of indifference to dental treatment.

Introducción: Los inadecuados hábitos de higiene bucal como la falta de cepillado dental, uso de irrigadores, pastas dentales fluoradas e hilo dental, así como la importancia de preocuparse por acudir al odontólogo ante cualquier problema en la cavidad oral y la continuidad del tratamiento son factores que podrían agravar el riesgo en los pacientes. Objetivo: Determinar la asociación entre el nivel de indiferencia al tratamiento dental y los hábitos de higiene bucal de los encuestados en adultos en edades comprendidas entre los 18 a 45 años de la urbanización Buenos Aires de Villa en Chorrillos, Perú. Material y Métodos: Se realizó un estudio observacional, analítico y transversal. El nivel de indiferencia al tratamiento dental fue evaluado mediante un cuestionario virtual traducido y posteriormente validado internamente (alpha de Cronbach: 0.91). Se tuvo un total de 249 participantes, entre ellos 150 hombres y 99 mujeres con una media edad de 30,69 años. Para la asociación de las variables cualitativas y el reporte de las Odds Ratio (OR) crudas y ajustadas, se empleó una regresión logística. Se trabajó con un nivel de significancia estadística de p<0.05 y un intervalo de confianza del 95%. Resultados: Se obtuvo un alto nivel de indiferencia al tratamiento dental en el 57.83% de los encuestados. Asimismo, se encontró una asociación estadísticamente significativa de la indiferencia al tratamiento dental con la educación (p= 0.012). Respecto a los hábitos de higiene bucal, se encontró asociación estadísticamente significativa con uso de hilo dental (OR=2.22; IC del 95%: 1.10-4.46) (p=0.025) y el cepillado antes de dormir (OR=5.26; IC del 95%: 2.26-12-22) (p<0.001). Conclusión: Se concluyó que existe una asociación estadísticamente significativa entre el nivel de indiferencia al tratamiento dental con los hábitos de higiene bucal uso de hilo dental y cepillado antes de dormir. Se aconseja realizar actividades en las comunidades para promover el cuidado de la salud bucal con la finalidad de reducir los niveles de indiferencia al tratamiento dental.

Targeting BPTF Sensitizes Pancreatic Ductal Adenocarcinoma to Chemotherapy by Repressing ABC-Transporters and Impairing Multidrug Resistance (MDR).

Pancreatic ductal adenocarcinoma (PDA) is characterized by an extremely poor prognosis due to its late diagnosis and strong chemoresistance to the current treatments. Therefore, finding new therapeutic targets is an urgent need nowadays. In this study, we report the role of the chromatin remodeler BPTF (Bromodomain PHD Finger Transcription Factor) as a therapeutic target in PDA. BPTF-silencing dramatically reduced cell proliferation and migration in vitro and in vivo in human and mouse PDA cell lines. Moreover, BPTF-silencing reduces the IC50 of gemcitabine in vitro and enhanced its therapeutic effect in vivo. Mechanistically, BPTF is required for c-MYC recruitment to the promoter of ABC-transporters and its downregulation facilitates gemcitabine accumulation in tumour cells, increases DNA damage, and a generates a strong synergistic effect in vivo. We show that BPTF is a therapeutic target in pancreatic ductal adenocarcinoma due to its strong effect on proliferation and in response to gemcitabine.

Bcl3 Couples Cancer Stem Cell Enrichment With Pancreatic Cancer Molecular Subtypes.

BACKGROUND & AIMS: The existence of different subtypes of pancreatic ductal adenocarcinoma (PDAC) and their correlation with patient outcome have shifted the emphasis on patient classification for better decision-making algorithms and personalized therapy. The contribution of mechanisms regulating the cancer stem cell (CSC) population in different subtypes remains unknown. METHODS: Using RNA-seq, we identified B-cell CLL/lymphoma 3 (BCL3), an atypical nf-κb signaling member, as differing in pancreatic CSCs. To determine the biological consequences of BCL3 silencing in vivo and in vitro, we generated bcl3-deficient preclinical mouse models as well as murine cell lines and correlated our findings with human cell lines, PDX models, and 2 independent patient cohorts. We assessed the correlation of bcl3 expression pattern with clinical parameters and subtypes. RESULTS: Bcl3 was significantly down-regulated in human CSCs. Recapitulating this phenotype in preclinical mouse models of PDAC via BCL3 genetic knockout enhanced tumor burden, metastasis, epithelial to mesenchymal transition, and reduced overall survival. Fluorescence-activated cell sorting analyses, together with oxygen consumption, sphere formation, and tumorigenicity assays, all indicated that BCL3 loss resulted in CSC compartment expansion promoting cellular dedifferentiation. Overexpression of BCL3 in human PDXs diminished tumor growth by significantly reducing the CSC population and promoting differentiation. Human PDACs with low BCL3 expression correlated with increased metastasis, and BCL3-negative tumors correlated with lower survival and nonclassical subtypes. CONCLUSIONS: We demonstrate that bcl3 impacts pancreatic carcinogenesis by restraining CSC expansion and by curtailing an aggressive and metastatic tumor burden in PDAC across species. Levels of BCL3 expression are a useful stratification marker for predicting subtype characterization in PDAC, thereby allowing for personalized therapeutic approaches.

Efectos de las Técnicas Manuales Orofaciales en la Disminución del Dolor Cervical. Una Revisión Bibliográfica / Effects of Orofacial Manual Techniques in the Reduction of Cervical Pain. A Bibliographic Review

Introducción: La prevalencia anual de dolor cervical oscila entre un 16,7% y un 75,1%, durando alrededor de 6 meses o más en el 14% de los casos, desencadenando en restricción del movimiento y dolor principalmente. Esto podría deberse a la relación entre la cabeza, columna cervical y estructuras relacionadas a la articulación temporomandibular (ATM). El objetivo de esta investigación es describir a través de la evidencia científica el efecto de las técnicas manuales orales en la disminución del dolor cervical. Métodos: Se incluyeron ensayos clínicos aleatorizados y revisiones sistemáticas encontrados en las bases de datos PubMed, Cochrane y PEDro. Realizando la extracción de datos a través de una tabla estructurada para cada tipo de estudio. Resultados: De 10 estudios seleccionados, 3 corresponden a revisiones sistemáticas y 7 a ensayos clínicos, donde la disminución del dolor cervical fue significativa al aplicar la terapia manual con ejercicios por sobre la terapia manual por sí sola. Por otro lado, las técnicas orofaciales fueron beneficiosas al combinarlas con terapia manual en comparación con un tratamiento habitual. No se encontraron diferencias significativas al aplicar ejercicios de Maitland y movilizaciones en comparación con ejercicios de flexibilidad, fortalecimiento, ejercicios de Mulligan y movilizaciones. Conclusión: Los resultados son debatibles, ya que la terapia manual por sí sola o combinada con ejercicios es la técnica más efectiva no solo en la disminución del dolor cervical, también en la mejoría de la calidad de vida.

Introduction: The annual prevalence of cervical pain ranges from 16.7% to 75.1%, lasting about 6 months or more in 14% of cases, triggering mainly movement restriction and pain. This could be due to the relationship between the head, cervical spine and structures related to the temporomandibular joint (TMJ). The objective of this research is to describe through scientific evidence the effect of oral techniques in reducing cervical pain. Methods: Randomized clinical trials and systematic reviews found in the PubMed, Cochrane and PEDro databases were included. Data extraction was done through a structured table for each type of study. Results: Out of 10 selected studies, 3 correspond to systematic reviews and 7 to clinical trials, where the decrease in neck pain was significant when applying manual therapy with exercises over manual therapy alone. On the other hand, orofacial techniques were beneficial when combined with manual therapy compared to an usual treatment. No significant differences were found when applying Maitland exercises and mobilizations compared to flexibility, strengthening, Mulligan exercises, and mobilizations (p<0.05). Conclusion: The results are debatable, as manual therapy alone or combined with exercises is the most effective technique not only in reducing neck pain, but also in improving quality of life.

Perennial halophyte Salicornia neei Lag.: Cell wall composition and functional properties of its biopolymers.

Salicornia neei halophyte extends in Argentina seashores. To envisage potential applications, cell wall sequential extraction performed on dry plant yielded 1.1, 2.4, 0.3 and 0.9% of pectin fractions respectively extracted by room temperature water, 90 °C-water, CDTA and Na2CO3. They contained 21-33% uronic acids (UA) with low degree of methylation and 0.5-1.2 M ratios of neutral sugars to UA. High arabinose level suggests that long arabinan side-chains maintain cell wall flexibility in water deficit. Fractions also contained 10-36% of proteins. The KOH-soluble fractions (4.3%) were mainly arabinoxylans. At 2.0% w/v, pectin fractions developed "weak gel"-type networks with Ca2+, while arabinoxylans generated "dilute solutions". Cellulose (28%) and lignin (45.1%) were the main biopolymers in the final residue, which showed low water swelling capacity (3.6 mL/g) due to lignin, increasing when arabinoxylans were also present. Phenolics (9.8%) were mainly water-extractable. Salicornia is a source of biopolymers and antioxidants potentially useful for food applications.

Exploiting oxidative phosphorylation to promote the stem and immunoevasive properties of pancreatic cancer stem cells.

Pancreatic ductal adenocarcinoma (PDAC), the fourth leading cause of cancer death, has a 5-year survival rate of approximately 7-9%. The ineffectiveness of anti-PDAC therapies is believed to be due to the existence of a subpopulation of tumor cells known as cancer stem cells (CSCs), which are functionally plastic, and have exclusive tumorigenic, chemoresistant and metastatic capacities. Herein, we describe a 2D in vitro system for long-term enrichment of pancreatic CSCs that is amenable to biological and CSC-specific studies. By changing the carbon source from glucose to galactose in vitro, we force PDAC cells to utilize OXPHOS, resulting in enrichment of CSCs defined by increased CSC biomarker and pluripotency gene expression, greater tumorigenic potential, induced but reversible quiescence, increased OXPHOS activity, enhanced invasiveness, and upregulated immune evasion properties. This CSC enrichment method can facilitate the discovery of new CSC-specific hallmarks for future development into targets for PDAC-based therapies.

ISG15 and ISGylation is required for pancreatic cancer stem cell mitophagy and metabolic plasticity.

Pancreatic cancer stem cells (PaCSCs) drive pancreatic cancer tumorigenesis, chemoresistance and metastasis. While eliminating this subpopulation of cells would theoretically result in tumor eradication, PaCSCs are extremely plastic and can successfully adapt to targeted therapies. In this study, we demonstrate that PaCSCs increase expression of interferon-stimulated gene 15 (ISG15) and protein ISGylation, which are essential for maintaining their metabolic plasticity. CRISPR-mediated ISG15 genomic editing reduces overall ISGylation, impairing PaCSCs self-renewal and their in vivo tumorigenic capacity. At the molecular level, ISG15 loss results in decreased mitochondrial ISGylation concomitant with increased accumulation of dysfunctional mitochondria, reduced oxidative phosphorylation (OXPHOS) and impaired mitophagy. Importantly, disruption in mitochondrial metabolism affects PaCSC metabolic plasticity, making them susceptible to prolonged inhibition with metformin in vivo. Thus, ISGylation is critical for optimal and efficient OXPHOS by ensuring the recycling of dysfunctional mitochondria, and when absent, a dysregulation in mitophagy occurs that negatively impacts PaCSC stemness.

Disaccharides obtained from carrageenans as potential antitumor agents.

Carrageenans are sulfated galactans found in certain red seaweeds with proven biological activities. In this work, we have prepared purified native and degraded κ-, ι-; and λ-carrageenans, including the disaccharides (carrabioses) and disaccharide-alditols (carrabiitols) from seaweed extracts as potential antitumor compounds and identified the active principle of the cytotoxic and potential antitumor properties of these compounds. Both κ and ι-carrageenan, as well as carrageenan oligosaccharides showed cytotoxic effect over LM2 tumor cells. Characterized disaccharides (carrabioses) and the reduced product carrabiitols, were also tested. Only carrabioses were cytotoxic, and among them, κ-carrabiose was the most effective, showing high cytotoxic properties, killing the cells through an apoptotic pathway. In addition, the cells surviving treatment with κ-carrabiose, showed a decreased metastatic ability in vitro, together with a decreased cell-cell and cell-matrix interactions, thus suggesting possible antitumor potential. Overall, our results indicate that most cytotoxic compounds derived from carrageenans have lower molecular weights and sulfate content. Potential applications of the results emerging from the present work include the use of disaccharide units such as carrabioses coupled to antineoplasics in order to improve its cytotoxicity and antimetastatic properties, and the use of ι-carrageenan as adjuvant or carrier in anticancer treatments.

Induction of DNA double-strand breaks and cellular senescence by human respiratory syncytial virus.

Human respiratory syncytial virus (HRSV) accounts for the majority of lower respiratory tract infections during infancy and childhood and is associated with significant morbidity and mortality. HRSV provokes a proliferation arrest and characteristic syncytia in cellular systems such as immortalized epithelial cells. We show here that HRSV induces the expression of DNA damage markers and proliferation arrest such as P-TP53, P-ATM, CDKN1A and γH2AFX in cultured cells secondary to the production of mitochondrial reactive oxygen species (ROS). The DNA damage foci contained γH2AFX and TP53BP1, indicative of double-strand breaks (DSBs) and could be reversed by antioxidant treatments such as N-Acetylcysteine (NAC) or reduced glutathione ethyl ester (GSHee). The damage observed is associated with the accumulation of senescent cells, displaying a canonical senescent phenotype in both mononuclear cells and syncytia. In addition, we show signs of DNA damage and aging such as γH2AFX and CDKN2A expression in the respiratory epithelia of infected mice long after viral clearance. Altogether, these results show that HRSV triggers a DNA damage-mediated cellular senescence program probably mediated by oxidative stress. The results also suggest that this program might contribute to the physiopathology of the infection, tissue remodeling and aging, and might be associated to long-term consequences of HRSV infections.

The EWS/FLI1 oncogenic protein inhibits expression of the Wnt inhibitor DICKKOPF-1 gene and antagonizes beta-catenin/TCF-mediated transcription.

Tumours of the Ewing family, which comprise Ewing's sarcoma and peripheral primitive neuroectodermal tumours, are highly aggressive and mostly affect children and adolescents. They are characterized by chromosomal translocations leading to the generation of fusion proteins between EWS (or very rarely FUS) and members of the E-twenty-six (ETS) family of transcription factors that are capable of transforming cells. EWS/FLI1, the most frequent fusion, is thought to cause transformation through activation or repression of specific target genes. We present evidence demonstrating that the Wnt inhibitor and beta-catenin/T-cell factor (TCF)-responsive gene DICKKOPF-1 (DKK-1) is a transcriptional target of EWS/FLI1, which can inhibit both basal and beta-catenin-induced transactivation of the DKK-1 promoter. Moreover, our data indicate that EWS/FLI1 has a more general effect on beta-catenin/TCF-mediated transcription since it can block transactivation of a consensus beta-catenin/TCF reporter construct. Consistently, Ewing tumour cells expressing different EWS/ETS translocations cannot engage beta-catenin/TCF-dependent transcription, whereas silencing of EWS/FLI1 restores beta-catenin responsiveness in A673 and RD-ES Ewing tumour cells. Accordingly, gene set enrichment analysis shows that beta-catenin/TCF target genes are significantly enriched among genes downregulated by EWS/FLI1 in the Ewing cell line A673. Mechanistically, the inhibitory effect of EWS/FLI1 can be overcome by a constitutively active TCF4 protein (TCF4-VP16). Moreover, EWS/FLI1 binds lymphoid enhancer factor 1, a TCF family member, and interferes with its binding to beta-catenin, which could explain its negative effect on beta-catenin/TCF-mediated transcription. Our results show that EWS/FLI1 inhibits both DKK-1 expression as well as beta-catenin/TCF-dependent transcription, which could contribute to progression of tumours of the Ewing family.

The Wnt antagonist DICKKOPF-1 gene is induced by 1alpha,25-dihydroxyvitamin D3 associated to the differentiation of human colon cancer cells.

The Wnt-beta-catenin pathway is aberrantly activated in most colon cancers. DICKKOPF-1 (DKK-1) gene encodes an extracellular Wnt inhibitor that blocks the formation of signalling receptor complexes at the plasma membrane. We report that 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3], the most active vitamin D metabolite, increases the level of DKK-1 RNA and protein in human SW480-ADH colon cancer cells. This effect is dose dependent, slow and depends on the presence of a transcription-competent nuclear vitamin D receptor (VDR). Accordingly, 1,25(OH)2D3 activates a 2300 bp fragment of the human DKK-1 gene promoter. Chromatin immunoprecipitation assays revealed that 1,25(OH)2D3 treatment induced a pattern of histone modifications which is compatible with transcriptionally active chromatin. DKK-1 is expressed at high level in colon cancer cell lines with a differentiated phenotype such as Caco-2 or HT-29. Exogenous expression of E-cadherin into SW480-ADH cells results in a strong adhesive phenotype and a 17-fold increase in DKK-1 RNA. In contrast, an E-cadherin blocking antibody inhibits 1,25(OH)2D3-induced differentiation of SW480-ADH cells and DKK-1 gene expression. Remarkably, in vivo treatment with the vitamin D analogue EB1089 induced DKK-1 protein expression in SW480-ADH cells xenografted in immunodeficient mice, and a correlation was observed in the expression of VDR and DKK-1 RNA in a series of 32 human colorectal tumours. These data indicate that 1,25(OH)2D3 activates the transcription of the DKK-1 gene, probably in an indirect way that is associated to the promotion of a differentiated phenotype. DKK-1 gene induction constitutes a novel mechanism of inhibition of Wnt signalling and antitumour action by 1,25(OH)2D3.