Binding Evolution of the Dengue Virus Envelope Against DC-SIGN: A Combined Approach of Phylogenetics and Molecular Dynamics Analyses Over 30 Years of Dengue Virus in Brazil.

The Red Queen Hypothesis (RQH), derived from Lewis Carroll's "Through the Looking-Glass", postulates that organisms must continually adapt in response to each other to maintain relative fitness. Within the context of host-pathogen interactions, the RQH implies an evolutionary arms race, wherein viruses evolve to exploit hosts and hosts evolve to resist viral invasion. This study delves into the dynamics of the RQH in the context of virus-cell interactions, specifically focusing on virus receptors and cell receptors. We observed multiple virus-host systems and noted patterns of co-evolution. As viruses evolved receptor-binding proteins to effectively engage with cell receptors, cells countered by altering their receptor genes. This ongoing mutual adaptation cycle has influenced the molecular intricacies of receptor-ligand interactions. Our data supports the RQH as a driving force behind the diversification and specialization of both viral and host cell receptors. Understanding this co-evolutionary dance offers insights into the unpredictability of emerging viral diseases and potential therapeutic interventions. Future research is crucial to dissect the nuanced molecular changes and the broader ecological consequences of this ever-evolving battle. Here, we combine phylogenetic inferences, structural modeling, and molecular dynamics analyses to describe the epidemiological characteristics of major Brazilian DENV strains that circulated from 1990 to 2022 from a combined perspective, thus providing us with a more detailed picture on the dynamics of such interactions over time.

Acidic Environment Induces Pyroptosis of NLRP3 Inflammatory Vesicle-Mediated Nucleus Pulposus Cells Through Up-Regulation of POSTN Expression

SUMMARY: Intervertebral disc degeneration (IVDD) is induced by nucleus pulposus (NP) dysfunction as a result of massive loss of NP cells. It has been reported that the acidic microenvironment of the intervertebral disc (IVD) can induce NP cell pyroptosis, and that up-regulation of periostin (POSTN) expression has a negative effect on NP cell survival. However, the relationship between the acidic environment, POSTN expression level and NP cell pyroptosis is unclear. Therefore, the aim of this study was to explore the relationship between acidic environment and POSTN expression level in NP cells, as well as the effect of POSTN in acidic environment on NP cell pyroptosis. NP cells were obtained from the lumbar vertebrae of Sprague Dawley (SD) male rats. These cells were divided into normal and acidic groups according to whether they were exposed to 6 mM lactic acid solution. And NP cells in the acidic group were additionally divided into three groups: (1) Blank group: no transfection; (2) NC group: cells transfected with empty vector plasmid; (3) sh-POSTN group: cells transfected with sh-POSTN plasmid to knock down the expression level of POSTN. Quantitative real-time PCR (qRT-PCR) and western blot was performed to assess the expression of POSTN at the mRNAand protein levels. CCK8 was used to evaluate cell survival. Western blot, in addition, was performed to examine acid-sensing ion channels (ASIC)-related proteins. And pyroptosis was detected by ELISA and western blot. The expression level of POSTN was significantly increased in NP cells in acidic environment. Knockdown of POSTN expression promoted the survival of NP cells in acidic environment and reduced the protein levels of ASIC3 and ASIC1a in NP cells. Moreover, knockdown of POSTN expression decreased the pyroptosis proportion of NP cells and the levels of pro-inflammatory cytokines interleukin (IL)-1β and IL-18. The levels of pyroptosis-related proteins NLRP3, ASC, cleaved-Caspase-1, and cleaved-GSDMD were also affected by the decreased POSTN expression. The extracellular acidic environment created by lactic acid solution activated NLRP3 inflammatory vesicle-induced caspase-1 to get involved in NP cell pyroptosis by up-regulating POSTN expression.

La degeneración del disco intervertebral (DDIV) es inducida por una disfunción del núcleo pulposo (NP) como resultado de una pérdida masiva de células NP. Se ha informado que el microambiente ácido del disco intervertebral (DIV) puede inducir la piroptosis de las células NP y que la regulación positiva de la expresión de periostina (POSTN) tiene un efecto negativo en la supervivencia de las células NP. Sin embargo, la relación entre el ambiente ácido, el nivel de expresión de POSTN y la piroptosis de las células NP es poco clara. Por lo tanto, el objetivo de este estudio fue explorar la relación entre el ambiente ácido y el nivel de expresión de POSTN en células NP, así como el efecto de POSTN en ambiente ácido sobre la piroptosis de las células NP. Las células NP se obtuvieron de las vertebras lumbares de ratas macho Sprague Dawley (SD). Estas células se dividieron en grupos normales y ácidos según se expusieron a una solución de ácido láctico 6 mM. Las células NP en el grupo ácido se dividieron adicionalmente en tres grupos: (1) Grupo en blanco: sin transfección; (2) grupo NC: células transfectadas con plásmido vector vacío; (3) grupo sh-POSTN: células transfectadas con plásmido sh-POSTN para reducir el nivel de expresión de POSTN. Se realizó una PCR cuantitativa en tiempo real (qRT-PCR) y una transferencia Western para evaluar la expresión de POSTN en los niveles de ARNm y proteína. Se utilizó CCK8 para evaluar la supervivencia celular. Además, se realizó una transferencia Western para examinar las proteínas relacionadas con los canales iónicos sensibles al ácido (ASIC). La piroptosis se detectó mediante ELISA y Western blot. El nivel de expresión de POSTN aumentó significativamente en células NP en ambiente ácido. La eliminación de la expresión de POSTN promovió la supervivencia de las células NP en un ambiente ácido y redujo los niveles de proteína de ASIC3 y ASIC1a en las células NP. Además, la eliminación de la expresión de POSTN disminuyó la proporción de piroptosis de las células NP y los niveles de citocinas proinflamatorias interleucina (IL) - 1β e IL-18. Los niveles de proteínas relacionadas con la piroptosis NLRP3, ASC, Caspasa-1 escindida y GSDMD escindida también se vieron afectados por la disminución de la expresión de POSTN. El ambiente ácido extracelular creado por la solución de ácido láctico activó la caspasa-1 inducida por vesículas inflamatorias NLRP3 para involucrarse en la piroptosis de las células NP mediante la regulación positiva de la expresión de POSTN.

A novel homozygous nonsense mutation in NECTIN4 gene in a Pakistani family with ectodermal dysplasia syndactyly syndrome 1.

BACKGROUND: Ectodermal dysplasia syndactyly syndrome 1 (EDSS1) is a rare hereditary disorder characterized by defects in teeth, hair, and nails in association with a fusion of the digits. Genetically, the disease phenotypes are caused by homozygous and compound heterozygous variants in NECTIN4 gene. OBJECTIVE: The main objective of the study was to identify the pathogenic sequence variant(s) for family screening and identification of carriers. METHODS: In the present study, the authors have investigated a large consanguineous family of Pakistani origin segregating autosomal recessive EDSS1. All the coding exons of the NECTIN4 gene were directly sequenced using gene-specific primers. RESULTS: The affected individuals presented the classical EDSS1 clinical features including sparse hair, hypoplastic nails with thick flat discolored nail plates, peg-shaped, conical, and widely spaced teeth with enamel hypoplasia, proximal cutaneous syndactyly of fingers and toes. Sequence analysis of the coding region of the NECTIN4 identified a novel nonsense variant [c.163C>T; p.(Arg55*)] in exon-2 of the gene. Computational analysis of protein structure revealed that the variant induced premature termination at Arg55 located in Ig-like V-loop region leading to loss of Ig-C2 type domains and transmembrane region, and most likely Nectin-4 function will be lost. STUDY LIMITATION: Gene expression studies are absent that would have strengthened the findings of computational analysis. CONCLUSION: The present study expanded the phenotypic and mutation spectrum of the NECTIN4 gene. Further, the study would assist in carrier testing and prenatal diagnosis of the affected families.

RIPOR2 Expression Decreased by HPV-16 E6 and E7 Oncoproteins: An Opportunity in the Search for Prognostic Biomarkers in Cervical Cancer.

High-risk human papillomavirus (HPV) infection is the main risk factor for cervical cancer (CC) development, where the continuous expression of E6 and E7 oncoproteins maintain the malignant phenotype. In Mexico, around 70% of CC cases are diagnosed in advanced stages, impacting the survival of patients. The aim of this work was to identify biomarkers affected by HPV-16 E6 and E7 oncoproteins that impact the prognosis of CC patients. Expression profiles dependent on E6 and E7 oncoproteins, as well as their relationship with biological processes and cellular signaling pathways, were analyzed in CC cells. A comparison among expression profiles of E6- and E7-expressing cells and that from a CC cohort obtained from The Cancer Genome Atlas (TCGA) demonstrated that the expression of 13 genes impacts the overall survival (OS). A multivariate analysis revealed that the downregulated expression of RIPOR2 was strongly associated with a worse OS. RIPOR2, including its transcriptional variants, were overwhelmingly depleted in E6- and E7-expressing cells. Finally, in a Mexican cohort, it was found that in premalignant cervical lesions, RIPOR2 expression decreases as the lesions progress; meanwhile, decreased RIPOR2 expression was also associated with a worse OS in CC patients.

Regulatory T-Cell Enhancement, Expression of Adhesion Molecules, and Production of Anti-Inflammatory Factors Are Differentially Modulated by Spheroid-Cultured Mesenchymal Stem Cells.

The culture of mesenchymal stem cells (MSCs) as spheroids promotes a more physiological cellular behavior, as it more accurately reflects the biological microenvironment. Nevertheless, mixed results have been found regarding the immunosuppressive properties of spheroid-cultured MSCs (3D-MSCs), the mechanisms of immunoregulation of 3D-MSCs being scarcely described at this point. In the present study, we constructed spheroids from MSCs and compared their immunosuppressive potential with that of MSCs cultured in monolayer (2D-MSCs). First, we evaluated the ability of 2D-MSCs and 3D-MSCs to control the activation and proliferation of T-cells. Next, we evaluated the percentage of regulatory T-cells (Tregs) after the co-culturing of peripheral blood mononuclear cells (PBMCs) with 2D-MSCs and 3D-MSCs. Finally, we investigated the expression of adhesion molecules, as well as the expressions of several anti-inflammatory transcripts in 2D-MSCs and 3D-MSCs maintained in both inflammatory and non-inflammatory conditions. Interestingly, our data show that several anti-inflammatory genes are up-regulated in 3D-MSCs, and that these cells can control T-cell proliferation. Nevertheless, 2D-MSCs are more efficient in suppressing the immune cell proliferation. Importantly, contrary to what was observed in 3D-MSCs, the expressions of ICAM-1 and VCAM-1 are significantly upregulated in 2D-MSCs exposed to an inflammatory environment. Furthermore, only 2D-MSCs are able to promote the enhancement of Tregs. Taken together, our data clearly show that the immunosuppressive potential of MSCs is significantly impacted by their shape, and highlights the important role of cell-cell adhesion molecules for optimal MSC immunomodulatory function.

Trophoblast cell surface antigen 2 expression predicts outcome in oral squamous cell carcinomas.

BACKGROUND: Trophoblast cell surface antigen 2 (TROP2) has unclear clinical role in oral squamous cell carcinomas (OSCC). Here, we investigated the association of TROP2 immunoexpression with clinicopathological parameters and survival of OSCC patients. SUBJECTS AND METHODS: Cancer-specific survival (CSS) and disease-free survival (DFS) were assessed in a cohort composed of 266 OSCC. An independent cohort with 88 OSCC samples matched with the normal oral tissue, as well as 17 metastatic lymph nodes, was used for validation. RESULTS: Multivariate analysis showed TROP2 as an independent marker of favorable prognosis for both CSS (HR: 0.60, 95% CI: 0.40-0.90, p = .01) and DFS (HR: 0.57, 95% CI: 0.36-0.89, p = .01). Furthermore, TROP2 protein expression was significantly higher in morphologically normal tissues compared to primary tumors (p < .0001) and lymph node metastases (p = .001), and it was significantly associated with CSS (HR: 0.26, 95% CI: 0.09-0.74, p = .008) in the validation cohort. A pooled mRNA analysis performed on the Oncomine™ database confirmed the underexpression in OSCC compared with normal tissues (p = .014). CONCLUSIONS: In summary, our results point to a favorable prognostic significance of TROP2 overexpression in a large cohort of oral cancer patients, suggesting it as an attractive clinical marker.

Association of IL-9, IL-10, and IL-17 Cytokines With Hepatic Fibrosis in Human Schistosoma mansoni Infection.

This is a case series study to evaluate immunological markers associated with schistosomiasis advanced fibrosis, including 69 patients from an endemic area from the State of Sergipe and from the Hepatology Service of the University Hospital in Sergipe, Brazil. Hepatic fibrosis was classified based on Niamey protocol for ultrasonography (US). Immune response to Schistosoma mansoni antigens was evaluated by stimulating peripheral blood mononuclear cells (PBMCs) from these patients with either adult worm (SWAP-10 µg/ml) or egg (SEA-10 µg/ml) antigens or purified protein derivative of turberculin (PPD-10 µg/ml) or phytohemagglutinin (PHA-1 µg/ml) for 72 h. The levels of IFN-γ, TNF-α, IL-5, IL-10, and IL-17 were measured in these supernatants by ELISA and IL-9 by Luminex. Single nucleotide polymorphisms in IL-17, IL10, and CD209 genes were genotyped using TaqMan probe by qPCR. Higher levels of IL-9, IL-10, and IL-17 were found in PBMC supernatants of patients with advanced hepatic fibrosis. Direct correlations were detected between IL-9 and IL-17 levels with US spleen sizes, portal vein diameters, and periportal thickening. The CD209 rs2287886 AG polymorphism patients produce higher IL-17 levels. Together, these data suggest a role of these cytokines in the immunopathogenesis of advanced fibrosis in human schistosomiasis.

Toxoplasma gondii-Induced Neutrophil Extracellular Traps Amplify the Innate and Adaptive Response.

Toxoplasmosis affects one-third of the human population worldwide. Humans are accidental hosts and are infected after consumption of undercooked meat and water contaminated with Toxoplasma gondii cysts and oocysts, respectively. Neutrophils have been shown to participate in the control of T. gondii infection in mice through a variety of effector mechanisms, such as reactive oxygen species (ROS) and neutrophil extracellular trap (NET) formation. However, few studies have demonstrated the role of neutrophils in individuals naturally infected with T. gondii. In the current study, we evaluated the activation status of neutrophils in individuals with acute or chronic toxoplasmosis and determined the role of T. gondii-induced NET formation in the amplification of the innate and adaptive immune responses. We observed that neutrophils are highly activated during acute infection through increased expression of CD66b. Moreover, neutrophils from healthy donors (HDs) cocultured with tachyzoites produced ROS and formed NETs, with the latter being dependent on glycolysis, succinate dehydrogenase, gasdermin D, and neutrophil elastase. Furthermore, we observed elevated levels of the chemokines (CXC motif) CXCL8 and (CC motif) CCL4 ligands in plasma from patients with acute toxoplasmosis and production by neutrophils from HDs exposed to T. gondii. Finally, we showed that T. gondii-induced NETs activate neutrophils and promote the recruitment of autologous CD4+ T cells and the production of interferon gamma (IFN-γ), tumor necrosis factor (TNF), interleukin 6 (IL-6), IL-17, and IL-10 by peripheral blood mononuclear cells. In conclusion, we demonstrated that T. gondii activates neutrophils and promotes the release of NETs, which amplify human innate and adaptive immune responses. IMPORTANCE Approximately one-third of the human population is estimated to be chronically infected with the obligate intracellular parasite Toxoplasma gondii. Humans are accidental hosts that are infected with T. gondii after consumption of undercooked meat or contaminated water. Neutrophils have been shown to control T. gondii growth by different mechanisms, including neutrophil extracellular traps (NETs). In the current study, we observed that neutrophils are highly activated during acute toxoplasmosis. We also determined that T. gondii-induced NETs are dependent on the energetic profile of neutrophils as well as the production of ROS and gasdermin D (GSDMD) cleavage. In addition, we showed that T. gondii-induced NETs activate neutrophils, promote the recruitment of autologous CD4+ T cells, and induce the production of cytokines by peripheral blood mononuclear cells, amplifying the innate and adaptive immune responses.

Association of Genetic Polymorphisms in DC-SIGN, Toll-Like Receptor 3, and Tumor Necrosis Factor α Genes and the Lewis-Negative Phenotype With Chikungunya Infection and Disease in Nicaragua.

BACKGROUND: Chikungunya infections range from subclinical infection to debilitating arthralgia and to chronic inflammatory rheumatism. Tumor necrosis factor (TNF) α, DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin), Toll-like receptor (TLR) 3, and blood groups have been directly or indirectly implicated in the susceptibility and pathogenesis of chikungunya. METHODS: To test the hypothesis that polymorphisms in genes coding for these molecules determine clinical outcomes of chikungunya infection, a retrospective case-control study was performed in León, Nicaragua. The study included 132 case patients and 132 controls, matched for age, sex and neighborhood. Case patients had clinical symptoms of chikungunya, which was diagnosed by means of polymerase chain reaction. Controls were individuals not reporting abrupt presentation of clinical chikungunya-like symptoms. Polymorphisms were identified by TaqMan single-nucleotide polymorphism genotyping assays. RESULTS: After adjustment for sociodemographic risk factors, chikungunya disease was associated with polymorphism in DC-SIGN and TLR3 genes (odds ratios, 5.2 and 3.3, respectively), and TNF-α with reduced persistent joint pain (0.24). Persistent joint pain was also associated with age, female sex and other comorbid conditions. Most interestingly, the Lewis-negative phenotype was strongly associated with both symptomatic chikungunya and immunoglobulin G seropositivity (odds ratios, 2.7, and 3.3, respectively). CONCLUSION: This study identified polymorphisms in DC-SIGN, TLR3, and TNF-α genes as well as Lewis-negative phenotype as risk factors for chikungunya infection and disease progression.

An epidermal growth factor motif of developmental endothelial locus 1 protein inhibits efficient angiogenesis in explanted squamous cell carcinoma in vivo

ABSTRACT Background: Cancer gene therapy using a nonviral vector is expected to be repeatable, safe, and inexpensive, and to have long-term effectiveness. Gene therapy using the E3 and C1 (E3C1) domain of developmental endothelial locus-1 (Del1) has been shown to improve prognosis in a mouse transplanted tumor model. Objective: In this study, we examined how this treatment affects angiogenesis in mouse transplanted tumors. Materials and methods: Mouse transplanted tumors (SCCKN human squamous carcinoma cell line) were injected locally with a nonviral plasmid vector encoding E3C1 weekly. Histochemical analysis of the transplanted tumors was then performed to assess the effects of E3C1 on prognosis. Results: All mice in the control group had died or reached an endpoint within 39 days. In contrast, one of ten mice in the E3C1 group had died by day 39, and eight of ten had died or reached an endpoint by day 120 (p < 0.01). Enhanced apoptosis in tumor stroma was seen on histochemical analyses, as was inhibited tumor angiogenesis in E3C1-treated mice. In addition, western blot analysis showed decreases in active Notch and HEY1 proteins. Conclusion: These findings indicate that cancer gene therapy using a nonviral vector encoding E3C1 significantly improved life-span by inhibiting tumor angiogenesis. (REV INVEST CLIN. 2021;73(1):39-51)

Testing the Stability and Validity of an Executive Dysfunction Classification Using Task-Based Assessment in Children and Adolescents.

OBJECTIVE: It is unclear if pediatric executive dysfunction assessed only with cognitive tasks predicts clinically relevant outcomes independently of psychiatric diagnoses. This study tested the stability and validity of a task-based classification of executive function. METHOD: A total of 2,207 individuals (6-17 years old) from the Brazilian High-Risk Cohort Study participated in this study (1,930 at baseline, 1,532 at follow-up). Executive function was measured using tests of working memory and inhibitory control. Dichotomized age- and sex-standardized performances were used as input in latent class analysis and receiver operating curves to create an executive dysfunction classification (EDC). The study tested EDC's stability over time, association with symptoms, functional impairment, a polymorphism in the CADM2 gene, polygenic risk scores (PRS), and brain structure. Analyses covaried for age, sex, social class, IQ, and psychiatric diagnoses. RESULTS: EDC at baseline predicted itself at follow-up (odds ratio [OR] = 5.11; 95% CI 3.41-7.64). Participants in the EDC reported symptoms spanning several domains of psychopathology and exhibited impairment in multiple settings, including more adverse school events (OR = 2.530; 95% CI 1.838-3.483). Children in the EDC presented higher attention-deficit/hyperactivity disorder and lower educational attainment PRS at baseline; higher schizophrenia PRS at follow-up; and lower chances of presenting a polymorphism in a gene previously linked to high performance in executive function (CADM2 gene). They also exhibited smaller intracranial volumes and smaller bilateral cortical surface areas in several brain regions. CONCLUSION: Task-based executive dysfunction is associated with several validators, independently of psychiatric diagnoses and intelligence. Further refinement of task-based assessments might generate clinically useful tools.

Human Bone Marrow Mesenchymal Stem/Stromal Cells Exposed to an Inflammatory Environment Increase the Expression of ICAM-1 and Release Microvesicles Enriched in This Adhesive Molecule: Analysis of the Participation of TNF-α and IFN-γ.

Bone marrow mesenchymal stem/stromal cells (BM-MSCs) have immunoregulatory capacity; therefore, they have been used in different clinical protocols in which it is necessary to decrease the immune response. This capacity is mainly regulated by TNF-α and IFN-γ, and it has been observed that cell-cell contact, mainly mediated by ICAM-1, is important for MSCs to carry out efficient immunoregulation. Therefore, in the present work, we analyzed the effect of TNF-α alone or in combination with IFN-γ on the expression of ICAM-1. Besides, given the importance of cell contact in the immunoregulatory function of MSCs, we analyzed whether these cells release ICAM-1+ microvesicles (MVs). Our results show for the first time that TNF-α is capable of increasing the early expression of ICAM-1 in human BM-MSCs. Also, we observed that TNF-α and IFN-γ have a synergistic effect on the increase in the expression of ICAM-1. Furthermore, we found that BM-MSCs exposed to an inflammatory environment release MVs enriched in ICAM-1 (MVs-ICAM-1high). The knowledge generated in this study will contribute to the improvement of in vitro conditioning protocols that favor the therapeutic effect of these cells or their products.

HPV Induces Changes in Innate Immune and Adhesion Molecule Markers in Cervical Mucosa With Potential Impact on HIV Infection.

While most HPV infections are asymptomatic and clear spontaneously, persistent infection with high-risk HPVs is associated with cervical cancer and with increased risk of HIV acquisition. Although several hypotheses have been proposed to explain this phenomenon, none has been confirmed. Our aim was to investigate the expression of host factors involved in the susceptibility to HIV infection among HPV-infected women. Cervical samples were collected to characterize the expression levels of HIV susceptibility markers in the mucosa of HPV-infected compared with HPV-uninfected women. No differences in the frequency of CCR5+, integrin α4ß7+, activated and memory CD4+ T-cell were detected between the groups. We additionally evaluated the expression levels of genes involved in innate immune responses and in cell adhesion. HPV infected patients expressed higher levels of TLR9 and lower levels of pattern recognition receptors that recognize RNA (TLR3, TLR7, and MDA5/IFIH1). We also detected an impaired IFN pathway, with an increased Type I IFN and a decreased IFNα2 receptor expression. HPV+ samples displayed reduced expression of genes for adherens and tight junctions. Taken together, these results suggest that although HPV infection does not result in the recruitment/activation of susceptible CD4+ T-cell in the female genital tract, it leads to changes in the innate antiviral immune responses and in cell adhesion that are likely to favor HIV infection.

An Epidermal Growth Factor Motif of Developmental Endothelial Locus 1 Protein Inhibits Efficient Angiogenesis in Explanted Squamous Cell Carcinoma In Vivo.

BACKGROUND: Cancer gene therapy using a nonviral vector is expected to be repeatable, safe, and inexpensive, and to have longterm effectiveness. Gene therapy using the E3 and C1 (E3C1) domain of developmental endothelial locus-1 (Del1) has been shown to improve prognosis in a mouse transplanted tumor model. OBJECTIVE: In this study, we examined how this treatment affects angiogenesis in mouse transplanted tumors. MATERIALS AND METHODS: Mouse transplanted tumors (SCCKN human squamous carcinoma cell line) were injected locally with a nonviral plasmid vector encoding E3C1 weekly. Histochemical analysis of the transplanted tumors was then performed to assess the effects of E3C1 on prognosis. RESULTS: All mice in the control group had died or reached an endpoint within 39 days. In contrast, one of ten mice in the E3C1 group had died by day 39, and eight of ten had died or reached an endpoint by day 120 (p < 0.01). Enhanced apoptosis in tumor stroma was seen on histochemical analyses, as was inhibited tumor angiogenesis in E3C1-treated mice. In addition, western blot analysis showed decreases in active Notch and HEY1 proteins. CONCLUSION: These findings indicate that cancer gene therapy using a nonviral vector encoding E3C1 significantly improved life-span by inhibiting tumor angiogenesis.

MiR-128 suppresses metastatic capacity by targeting metadherin in breast cancer cells.

BACKGROUND: Breast cancer, the most common cancer in women worldwide, causes the vast majority of cancer-related deaths. Undoubtedly, tumor metastasis and recurrence are responsible for more than 90 percent of these deaths. MicroRNAs are endogenous noncoding RNAs that have been integrated into almost all the physiological and pathological processes, including metastasis. In the present study, the role of miR-128 in breast cancer was investigated. RESULTS: Compared to the corresponding adjacent normal tissue, the expression of miR-128 was significantly suppressed in human breast cancer specimens. More importantly, its expression level was reversely correlated to histological grade of the cancer. Ectopic expression of miR-128 in the aggressive breast cancer cell line MDA-MB-231 could inhibit cell motility and invasive capacity remarkably. Afterwards, Metadherin (MTDH), also known as AEG-1 (Astrocyte Elevated Gene 1) and Lyric that implicated in various aspects of cancer progression and metastasis, was further identified as a direct target gene of miR-128 and its expression level was up-regulated in clinical samples as expected. Moreover, knockdown of MTDH in MDA-MB-231 cells obviously impaired the migration and invasion capabilities, whereas re-expression of MTDH abrogated the suppressive effect caused by miR-128. CONCLUSIONS: Overall, these findings demonstrate that miR-128 could serve as a novel biomarker for breast cancer metastasis and a potent target for treatment in the future.

Expression of DC-SIGN-like C-Type Lectin Receptors in Salmo salar.

C-Type Lectin Receptors (CTLR) are involved in the activation of innate and adaptative immune responses. Among these receptors, the Dendritic Cell-Specific ICAM-3-Grabbing nonintegrin (DC-SIGN/CD209) has become a hot topic due to its ability to bind and facilitate the infections processes of several pathogens. Although well characterized in mammals, little documentation exists about the receptor in salmonid fishes. Here, we report the sequence and expression analysis of eight DC-SIGN-like genes in Salmo salar. Each receptor displays structural similarities to DC-SIGN molecules described in mammals, including internalization motifs, a neck region with heptad repeats, and a Ca+2-dependent carbohydrate recognition domain. The receptors are expressed in multiple tissues of fish, and fish cell lines, with differential expression upon infection with viral and bacterial pathogens. The identification of DC-SIGN-like receptors in Salmo salar provides new information regarding the structure of the immune system of salmon, potential markers for cell subsets, as well as insights into DC-SIGN conservation across species.

Biomarkers in vesicoureteral reflux: an overview.

Aim: This article aimed to review the role of cytokines, chemokines, growth factors and cellular adhesion molecules as biomarkers for vesicoureteral reflux (VUR) and reflux nephropathy (RN). Methods: We reviewed articles from 1979 onward by searching PubMed and Scopus utilizing the combination of words: 'VUR' or 'RN' and each one of the biomarkers. Results: Genetic, inflammatory, fibrogenic, environmental and epigenetic factors responsible for renal scarring need to be better understood. TGF-ß, IL-10, IL-6, IL-8 and TNF seem to exert a role in VUR particularly in RN based on the current literature. Serum levels of procalcitonin have been also associated with high-grade VUR and RN. These molecules should be more intensively evaluated as potential biomarkers for renal scarring in VUR. Conclusion: Further studies are necessary to define which molecules will really be of utility in clinical decisions and as therapeutic targets for VUR and RN.

Genomic characterization and prognostication applied to a Brazilian cohort of patients with myelofibrosis.

Genomic characterization of patients with myeloproliferative neoplasms (MPN) may lead to better diagnostic classification, prognostic assessment, and treatment decisions. These goals are particularly important in myelofibrosis (MF). We performed target Next Generation Sequencing for a panel of 255 genes and Chromosome Microarray Analysis (CMA) in 27 patients with MF. Patients were classified according to genomic findings and we compared the performance of a personalized prognostication system with IPSS, MIPSS70 and MIPSS70 + v2. Twenty-six patients presented mutations: 11.1% had single driver mutations in either JAK2, CALR or MPL; 85.2% had mutations in non-restricted genes (median: 2 per patient). CMA was abnormal in 91.7% of the 24 cases with available data. Copy-Number-Neutral Loss-of-Heterozygosity was the most common finding (66.7%). Del13q was the most frequent copy number variation, and we could define a 2.4 Mb minimally affected region encompassing RB1, SUCLA2 and CLLS2 loci. The largest genomic subgroup consisted of patients with mutations in genes involved with chromatin organization and splicing control (40.7%) and the personalized system showed better concordance and accuracy than the other prognostic systems. Comprehensive genomic characterization reveals the striking genetic complexity of MF and, when combined with clinical data, led, in our cohort, to better prognostication performance.

MIC4 from Toxoplasma gondii: A Lectin Acting as a Toll-Like Receptor Agonist.

Tachyzoites, which are infective forms of Toxoplasma gondii, use their actinomyosin system to move over surfaces and invade host cells. Central to this process is the regulated release of micronemes organelles contents. The microneme protein 4 (MIC4) has the property to recognize galactosides residues linked to glycoproteins on the host cell surface. This property allows that MIC4 binds to TLR2- and TLR4 N-linked glycans and promote the activation of cell innate immune cells and secretion of inflammatory cytokines, acting on resistance against the parasite. Obtention of MIC4 from T. gondii requires several purification steps, is time-consuming and provides low yield. Therefore, this section details the protocol for prokaryotic expression, production, and purification of recombinant MIC4 (rMIC4) and for experimental assays to confirm its biological activity.

Production and Characterization of MIC1: A Lectin from Toxoplasma gondii.

Some lectins of pathogens interact with host cells through the recognition of specific carbohydrates displayed on the mammals' cell surface. The microneme protein 1 (MIC1) from Toxoplasma gondii has a lectin domain that specifically binds sialic acid residues, often found in the terminal positions of N-glycans of mammalian cells. The necessary studies on the MIC1 biological roles have been limited initially by the laborious purification of the protein from T. gondii tachyzoites and the low yields verified. Then Escherichia coli has been transformed with a construct containing the MIC1 gene, and the obtained recombinant MIC1 (rMIC1) has been purified from the inclusion bodies. Herein, we detail the methodology of heterologous production and purification of rMIC1 and protocols to assay the rMIC1 lectin ability.