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Targeting non-coding RNAs (ncRNAs), including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), has recently emerged as a promising strategy for treating malignancies and other diseases. In recent years, the development of ncRNA-based therapeutics for targeting protein-coding and non-coding genes has also gained momentum. This review systematically examines ongoing and completed clinical trials to provide a comprehensive overview of the emerging landscape of ncRNA-based therapeutics. Significant efforts have been made to advance ncRNA therapeutics to early clinical studies. The most advanced trials have been conducted with small interfering RNAs (siRNAs), miRNA replacement using nanovector-entrapped miRNA mimics, or miRNA silencing by antisense oligonucleotides. While siRNA-based therapeutics have already received FDA approval, miRNA mimics, inhibitors, and lncRNA-based therapeutics are still under evaluation in preclinical and early clinical studies. We critically discuss the rationale and methodologies of ncRNA targeting strategies to illustrate this rapidly evolving field.
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Ensaios Clínicos como Assunto , Neoplasias , RNA não Traduzido , Humanos , Neoplasias/genética , Neoplasias/terapia , RNA não Traduzido/genética , RNA não Traduzido/uso terapêutico , MicroRNAs/genética , MicroRNAs/uso terapêutico , MicroRNAs/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Animais , RNA Interferente Pequeno/uso terapêuticoRESUMO
BACKGROUND: To assess how the diagnostic reproducibility of the 2018 Classification of Gingival Recession Defects (GRD) could be applied when comparing in-person chairside measurements with photographic measurements. METHODS: Thirty-four GRD were photographed and evaluated by 4 masked operators. For each case, the operators measured twice recession type (RT), recession depth (RD), keratinized tissue width (KTW), gingival thickness (GT), detectability of the cemento-enamel junction (CEJ), and presence of root steps (RSs), chairside, and on photographs. Intraclass correlation coefficient (ICC) with 95% confidence intervals (CI) was calculated for RD and KTW; Kappa with 95% CI was used for GT, CEJ, and RS; quadratic weighted Kappa with 95% CI was used for RT. RESULTS: RD, KTW, and RT showed excellent overall intra-operator agreement (> 0.93), and from good to excellent overall inter-operator agreement (> 0.80), for both clinical and photographic measurements. Agreements were lower for GT, CEJ, and RS. Overall clinical and photographic agreements were within 0.1 difference for every variable, except for inter-operator agreement for RS which was 0.72 for clinical measurements and 0.45 for photographic measurements. The lowest overall agreement between clinical versus photographic measurements existed for CEJ (0.28) and RS (0.35). CONCLUSIONS: Variables composing the 2018 Classification of GRD are reproducible, both clinically and on photographs, with comparable agreements. The overall agreement was higher for KTW, RD, and RT, and lower for GT, CEJ, and RS, for both clinical and photographic measurements. The comparison between chairside and photographic evaluations indicated fair to excellent agreement for most variables, with CEJ and RS showing fair agreement. PLAIN LANGUAGE SUMMARY: As digital diagnostics evolve to facilitate clinical diagnostic measurement, we aimed to assess the effectiveness of intraoral photography for diagnosing gingival recession defects (GRD) according to the 2018 Classification of GRD, compared to traditional clinical examination. Standardized photographs of thirty-four GRD cases were captured. Four masked operators evaluated the same gingival recessions twice in a clinical setting and twice using photographs. Measurement repeatability within and between operators was calculated for both clinical and photographic settings, and the two settings were compared. Continuous measurements such as recession depth and keratinized tissue width, as well as the evaluation of interproximal attachment height (recession type), showed excellent agreement both clinically and photographically. Agreement was lower for gingival thickness and the detectability of tooth anatomical landmarks, such as the cemento-enamel junction and the presence of root steps. Overall, the agreement between chairside and photographic evaluations was generally good, but lower when evaluating tooth anatomical landmarks. The variables composing the 2018 Classification of GRD are reproducible in both clinical and photographic settings, with comparable levels of agreement. However, there was consistently worse agreement for gingival thickness and when evaluating tooth anatomical landmarks.
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LNA-i-miR-221 is a novel microRNA(miRNA)-221 inhibitor designed for the treatment of human malignancies. It has recently undergone phase 1 clinical trial (P1CT) and early pharmacokinetics (PKs) data in cancer patients are now available. We previously used multiple allometric interspecies scaling methods to draw inferences about LNA-i-miR-221 PKs in humans and estimated the patient dose based on the safe and pharmacodynamic (PD) active dose observed in mice, therefore providing a framework for the definition of safe starting and escalation doses for the P1CT. The preliminary data collected during the P1CT showed that the LNA-i-miR-221 anticipated doses, according to our human PK estimation approach, were indeed well tolerated and effective. PD data demonstrated concentration-dependent downregulation of miR-221 and upregulation of its CDKN1B/p27 and PTEN canonical targets as well as stable disease in 8 (50.0%) patients and partial response in 1 (6.3%) colorectal cancer case. Here, we detail the experimentally evaluated PK parameters of LNA-i-miR-221 in human, using both a non-compartmental and a population PKs approach. The population approach was adequately described by a three-compartments model with first-order elimination. The recorded age, sex and body weight of patients were evaluated as potential covariates. The estimated typical population parameter values were clearance (CL = 200 mL/h/kg), central volume of distribution (V1 = 45 mL/kg), peripheral volume of distribution (V2 = 200 mL/kg, volume of the second peripheral compartment V3 = 930 mL/h/kg) and inter-compartmental clearance (Q2 = 480 mL/h/kg and Q3 = 68 mL/h/kg). Age was found to be a predictor of Q3, with a statistically significant correlation. This work aimed also at retrospectively comparing the measured plasmatic clearance values with those predicted by different allometric scaling approaches. Our comparative analysis showed that the most accurate prediction was achieved by applying the single species allometric scaling approach and that the use of more than one species in allometric scaling to predict therapeutic oligonucleotides PKs would not necessarily generate the best prediction. Finally, our predictive approach was found accurate not only in predicting the main PK parameters in human but suggesting the range of effective and safe dose to be applied in the next clinic phase 2.
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A 45-year-old female patient presented with a lack of inter-implant papilla after a partially edentulous anterior area was rehabilitated with dental implants. The soft tissue phenotype and inter-implant papilla was augmented using the 'iceberg' connective tissue graft, followed by a second surgical procedure where a strip gingival graft was combined with a connective tissue graft inserted underneath a pouch prepared into the previous 'iceberg' connective tissue graft at the level of the crest ('garage' approach), further enhancing soft tissue volume in that region. This technique aims to improve mucosal thickness and supracrestal tissue height while addressing esthetic concerns associated with multiple implant placements in the anterior region. The final esthetic outcome was excellent, harmonious soft tissue with appropriate thickness, symmetry with adjacent teeth, well-shaped interdental and inter-implant papilla with high patient satisfaction, making this approach a valuable addition to a surgeon's armamentarium. Future clinical studies are needed to evaluate the performance of this novel approach.
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AIM: To compare the efficacy of non-surgical re-instrumentation (NSR) and papillary preservation flap (PPF) surgery at single-rooted teeth with residual pockets. MATERIALS AND METHODS: Patients with at least a residual pocket depth (PD ≥ 5 mm) after Steps I and II were enrolled and randomly assigned to receive NSR or PPF surgery. The primary outcome was PD reduction, and secondary outcomes were clinical attachment level (CAL) change and patient-reported outcome measures (PROMs). Outcome variables were measured at baseline, 3 and 6 months. The examiner was blinded. Statistical analysis, one site for each patient, included descriptive statistics and analysis of covariance. RESULTS: Forty-six participants were enrolled, and one patient dropped out in the PPF group. After 6 months, both treatments resulted in significant PD reduction (1.3 ± 1.2 mm, p = .009 NSR; 2.0 ± 0.7 mm, p < .001 PPF) and CAL gain (1.0 ± 2.4 mm, p = .031 NSR; 1.4 ± 0.8 mm, p < .001 PPF). PD reduction between groups was not statistically significant (diff: 0.6 mm; 95% confidence interval [CI] [-0.3 to 1.5]; p = .167). Pocket closure was 61% NSR versus 86% PPF (p = .091). Smoking was associated with less PD reduction of almost 1 mm in both treatments. Treatment time was longer for PPF surgery, but PROMs and post-operative pain were similar between groups. CONCLUSIONS: Both NSR and PPF reduced PD without significant difference between treatments at 6 months. PPF surgery may offer faster PD reduction, but smoking habits reduce treatment efficacy.
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Colorectal cancer (CRC) is one of the most common malignancies and a relevant cause of cancer-related deaths worldwide. Dysregulation of microRNA (miRNA) expression has been associated with the development and progression of various cancers, including CRC. Among them, miR-221 emerged as an oncogenic driver, whose high expression is associated with poor patient prognosis. The present study was conceived to investigate the anti-CRC activity of miR-221 silencing based on early clinical data achieved from a first-in-human study by our group. Going back from bedside to bench, we demonstrated that LNA-i-miR-221 reduces cell viability, induces apoptosis in vitro, and impairs tumor growth in preclinical in vivo models of CRC. Importantly, we disclosed that miR-221 directly targets TP53BP2, which, together with TP53INP1, is known as a positive regulator of the TP53 apoptotic pathway. We found that (1) both these genes are overexpressed following miR-221 inhibition, (2) the strong anti-tumor activity of LNA-i-miR-221 was selectively observed on TP53 wild-type cells, and (3) this activity was reduced in the presence of the TP53-inhibitor Pifitrin-α. Our data pave the way to further investigations on TP53 functionality as a marker predictive of response to miR-221 silencing, which might be relevant for clinical applications.
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The aim of this systematic review (SR) was to assess whether tooth mobility (TM) increases the risk of tooth extraction/loss. The protocol was registered in PROSPERO database (CRD42023485425). The focused PECO questions were as follows: (1) "In patients with periodontitis, undergoing periodontal treatment, are teeth affected by mobility at higher risk of being extracted/lost compared to non-mobile teeth, with a minimum follow-up of 10 years?" and (2) "In these patients, does varying degrees of tooth mobility increase the risk of tooth extraction/loss, with a minimum follow-up of 10 years?". Results were reported according to PRISMA statement. Electronic and manual searches were conducted to identify longitudinal studies. The different assessments of tooth mobility were pooled into three groups: TM0: Undetectable tooth mobility, TM1: Horizontal/Mesio-distal mobility ≤1 mm, TM2: Horizontal/Mesio-distal mobility >1 mm or vertical tooth mobility. Tooth loss was the primary outcome. Various meta-analyses were conducted, including subgroup analyses considering different follow-up lengths and the timing of TM assessment, along with sensitivity analyses. A trial sequential analysis was also performed. Eleven studies were included (1883 patients). The mean follow-up range was 10-25 years. The weighted total of included teeth, based on the sample size, was 18 918, with a total of 1604 (8.47%) extracted/lost teeth. The overall rate of tooth extraction/loss increased with increasing mobility: TM0 was associated with a 5.85% rate (866/14822), TM1 with the 11.8% (384/3255), TM2 with the 40.3% (339/841). Mobile teeth (TM1/TM2) were at an increased risk for tooth extraction/loss, compared to TM0 (HR: 2.85; [95% CI 1.88-4.32]; p < .00001). TM1 had a higher risk than TM0 (HR: 1.96; [95% CI 1.09-3.53]; p < .00001). TM2 had a higher risk than TM1 (HR: 2.85; [95% CI 2.19-3.70]; p < .00001) and TM0 (HR: 7.12; [95% CI 3.27-15.51]; p < .00001). The results of the tests for subgroup differences were not significant. Sensitivity meta-analyses yielded consistent results with other meta-analyses. Within the limits of the quality of the studies included in the meta-analyses, mobile teeth were at higher risk of being extracted/lost in the long-term and higher degrees of TM significantly influenced clinicians' decision to extract a tooth. However, most teeth can be retained in the long-term and thus TM should not be considered a reason for extraction or a risk factor for tooth loss, regardless of the degree of TM.
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BACKGROUND: Long-term survival induced by anticancer treatments discloses emerging frailty among breast cancer (BC) survivors. Trastuzumab-induced cardiotoxicity (TIC) is reported in at least 5% of HER2+BC patients. However, TIC mechanism remains unclear and predictive genetic biomarkers are still lacking. Interaction between systemic inflammation, cytokine release and ADME genes in cancer patients might contribute to explain mechanisms underlying individual susceptibility to TIC and drug response variability. We present a single institution case series to investigate the potential role of genetic variants in ADME genes in HER2+BC patients TIC experienced. METHODS: We selected data related to 40 HER2+ BC patients undergone to DMET genotyping of ADME constitutive variant profiling, with the aim to prospectively explore their potential role in developing TIC. Only 3 patients ("case series"), who experienced TIC, were compared to 37 "control group" matched patients cardiotoxicity-sparing. All patients underwent to left ventricular ejection fraction (LVEF) evaluation at diagnosis and during anti-HER2 therapy. Each single probe was clustered to detect SNPs related to cardiotoxicity. RESULTS: In this retrospective analysis, our 3 cases were homogeneous in terms of clinical-pathological characteristics, trastuzumab-based treatment and LVEF decline. We identified 9 polymorphic variants in 8 ADME genes (UGT1A1, UGT1A6, UGT1A7, UGT2B15, SLC22A1, CYP3A5, ABCC4, CYP2D6) potentially associated with TIC. CONCLUSION: Real-world TIC incidence is higher compared to randomized clinical trials and biomarkers with potential predictive value aren't available. Our preliminary data, as proof of concept, could suggest a predictive role of pharmacogenomic approach in the identification of cardiotoxicity risk biomarkers for anti-HER2 treatment.
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Neoplasias da Mama , Cardiotoxicidade , Polimorfismo de Nucleotídeo Único , Trastuzumab , Humanos , Feminino , Trastuzumab/efeitos adversos , Trastuzumab/farmacocinética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Cardiotoxicidade/genética , Pessoa de Meia-Idade , Estudos Retrospectivos , Antineoplásicos Imunológicos/efeitos adversos , Antineoplásicos Imunológicos/farmacocinética , Idoso , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , AdultoRESUMO
Polynucleotides and Hyaluronic Acid (PN-HA) mixture showed several effects in modulation of healing process. The aim of this study was to assess the safety and clinical performance of PN-HA alone or in association with Deproteinized Bovine Bone Mineral (DBBM) with papillary preservation flaps (PPF) in the treatment of residual pockets. A total of 43 patients with 55 infra-bony defects were recruited; 30% were smokers. The mean baseline Probing Depth (PD) was 7.7 ±1.9 mm with a corresponding mean recession (Rec) of 1.9± 1.3 mm. The depth of infra-bony defect at the surgical measurement was 5.2±2.1 mm. DBBM was applied at 56% of the defects considered as not-containing based on clinical judgment. Healing was uneventful at all sites. After one year, PD reduction was 4.4±1.8 mm with a Rec increase of 1.0 ±0.8 mm. Detected bone fill at x-ray was 3.5 ± 1.9mm. The multilevel analysis showed that absence of smoking habits was associated with improved PD reduction (P =0.026) and bone gain (P= 0.039). PN-HA mixture is a safe product for periodontal surgery and seems to promote clinical benefit in the treatment of residual pockets associated to infra-bony defects.
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Mast cells localize to mucosal tissues and contribute to innate immune defense against infection. How mast cells sense, differentiate between, and respond to bacterial pathogens remains a topic of ongoing debate. Using the prototype enteropathogen Salmonella Typhimurium (S.Tm) and other related enterobacteria, here we show that mast cells can regulate their cytokine secretion response to distinguish between extracellular and invasive bacterial infection. Tissue-invasive S.Tm and mast cells colocalize in the mouse gut during acute Salmonella infection. Toll-like Receptor 4 (TLR4) sensing of extracellular S.Tm, or pure lipopolysaccharide, causes a modest induction of cytokine transcripts and proteins, including IL-6, IL-13, and TNF. By contrast, type-III-secretion-system-1 (TTSS-1)-dependent S.Tm invasion of both mouse and human mast cells triggers rapid and potent inflammatory gene expression and >100-fold elevated cytokine secretion. The S.Tm TTSS-1 effectors SopB, SopE, and SopE2 here elicit a second activation signal, including Akt phosphorylation downstream of effector translocation, which combines with TLR activation to drive the full-blown mast cell response. Supernatants from S.Tm-infected mast cells boost macrophage survival and maturation from bone-marrow progenitors. Taken together, this study shows that mast cells can differentiate between extracellular and host-cell invasive enterobacteria via a two-step activation mechanism and tune their inflammatory output accordingly.
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Infecções por Enterobacteriaceae , Infecções por Salmonella , Camundongos , Animais , Humanos , Mastócitos , Infecções por Salmonella/microbiologia , Salmonella typhimurium/genética , Citocinas/metabolismoRESUMO
UMG1 is a unique epitope of CD43, not expressed by normal cells and tissues of haematopoietic and non-haematopoietic origin, except thymocytes and a minority (<5%) of peripheral blood T lymphocytes. By immunohistochemistry analysis of tissue microarray and pathology slides, we found high UMG1 expression in 20%-24% of diffuse large B-cell lymphomas (DLBCLs), including highly aggressive BCL2high and CD20low cases. UMG1 membrane expression was also found in DLBCL bone marrow-infiltrating cells and established cell lines. Targeting UMG1 with a novel asymmetric UMG1/CD3ε-bispecific T-cell engager (BTCE) induced redirected cytotoxicity against DLBCL cells and was synergistic with lenalidomide. We conclude that UMG1/CD3ε-BTCE is a promising therapeutic for DLBCLs.
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Linfoma Difuso de Grandes Células B , Linfócitos T , Humanos , Linfócitos T/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Imuno-HistoquímicaRESUMO
OBJECTIVE: To evaluate the impact of COVID-19 pandemic among a sample of Italian dentists in terms of infection, strategies for infection control, organization of the dental clinic, attitude, and behavior. MATERIAL AND METHODS: This was a cross-sectional survey. The sample consisted of 8000 Italian dentists selected among 63,375 using a computerized random sampling method. An electronic informed consent had to be signed. The questionnaire categories were on demographic, infection risk management, organization, and dentists' attitude and behavior. Geographic macro-areas were used for subgroup analysis. RESULTS: Among 8000 invited dentists, 2443 agreed to participate to the survey (30.6%). Mean age was 51.2 years, women were 34.5%. A total of 6.1% self-reported COVID-19 experience and higher rate of infection was reported in north Italy compared to the south (p < 0.05). FFP2/FFP3 respirators (97.1%) and visors (97.4%) were used by almost all dentists. While, natural ventilation and mouthwashes were the most frequent approaches used to reduce the infection risk. Most of the dentists reported positive attitude, nevertheless 83.6% felt an increased responsibility. CONCLUSION: The self-reported COVID-19 prevalence was 6.1% with some differences among geographic areas. COVID 19 had a deep impact on preventive strategies, dental office organization, and behavior within this sample.
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BACKGROUND: Asthma is a clinical syndrome characterized by recurrent episodes of airway obstruction, bronchial hyperresponsiveness and airway inflammation. Most patients with asthma present a "type 2" (TH2) inflammation. ILC2 and TH2 cells release cytokines IL4, IL-13 and IL-5. CRSwNP is a condition characterized by hyposmia or anosmia, nasal congestion, nasal discharge, and face pain or pressure that last for at least 12 weeks in a row without relief. Both asthma and CRSwNP are often characterized by a type 2 inflammation endotype and are often present in the same patient. Dupilumab is a fully human monoclonal antibody targeting the interleukin-4 receptor α (IL-4Rα) subunit, blocking IL4/IL-4Rα binding and IL13. It has been labelled for the treatment of moderate to severe asthma in patients from the age of 12 years with an eosinophilic phenotype, and it has demonstrated efficacy and acceptable safety. Our study aims to investigate the effects of dupilumab on type 2 inflammatory biomarkers, such as eosinophils and eosinophil cationic protein (ECP). ECP is an eosinophil-derived substance contained in granules that are released during inflammation and causes various biological effects, including tissue damage in asthmatic airways. METHODS: ECP, Eosinophil counts (EOS), and total immunoglobulin E (IgE) levels were longitudinally measured using immunoassays in the serum of 21 patients affected by CRSwNP, of which 17 had asthma as a comorbidity, receiving 300 mg dupilumab every two weeks. RESULTS: The EOS and ECP, after a first phase of significant increase due to the intrinsic characteristic of the block of IL-4 and IL-13, returned to the baseline 10 months after the initial administration of dupilumab. Fractional exhaled nitric oxide (FeNO) and serum total IgE decreased significantly after 9 months. Asthma Control Test (ACT) scores improved after dupilumab treatment. FEV1% and FEV1 absolute registered a significant improvement at 10 months. CONCLUSIONS: Patients who received 300 milligrams of dupilumab every two weeks first experienced a temporary increase in eosinophils (EOS) and eosinophil cationic protein (ECP), then exhibited a gradual decline in these variables with a subsequent return to the initial baseline levels. When compared to the baseline, we observed that the levels of IgE and FeNO decreased over time, while there was an increase in both FEV1 and FEV1%.
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Diabetes mellitus (DM) is a complex and multifactorial disease characterised by high blood glucose. Type 2 Diabetes (T2D), the most frequent clinical condition accounting for about 90% of all DM cases worldwide, is a chronic disease with slow development usually affecting middle-aged or elderly individuals. T2D represents a significant problem of public health today because its incidence is constantly growing among both children and adults. It is also estimated that underdiagnosis prevalence would strongly further increase the real incidence of the disease, with about half of T2D patients being undiagnosed. Therefore, it is important to increase diagnosis accuracy. The current interest in RNA molecules (both protein- and non-protein-coding) as potential biomarkers for diagnosis, prognosis, and treatment lies in the ease and low cost of isolation and quantification with basic molecular biology techniques. In the present study, we analysed the transcriptome in serum samples collected from T2D patients and unaffected individuals to identify potential RNA-based biomarkers. Microarray-based profiling and subsequent validation using Real-Time PCR identified an uncharacterised long non-coding RNA (lncRNA) transcribed from the ASAP1 locus as a potential diagnostic biomarker. ROC curve analysis showed that a molecular signature including the lncRNA and the clinicopathological parameters of T2D patients as well as unaffected individuals showed a better diagnostic performance compared with the glycated haemoglobin test (HbA1c). This result suggests that the application of this biomarker in clinical practice would help to improve the diagnosis, and therefore the clinical management, of T2D patients. The proposed biomarker would be useful in the context of predictive, preventive, and personalised medicine (3PM/PPPM).
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Diabetes Mellitus Tipo 2 , Hiperglicemia , RNA Longo não Codificante , Adulto , Idoso , Criança , Humanos , Pessoa de Meia-Idade , Diabetes Mellitus Tipo 2/genética , Saúde Pública , RNA Longo não Codificante/genéticaRESUMO
The main aim of this paper is to discuss the socio-political meaning of the transnational literary production made by female migrant writers. Thus, it analyses their role in the framework of the 'hybrid' literary production of the 21st century in Europe, such as Spain and Italy. Moving away from the idea of national literatures, this paper investigates literature as a geographical and emotional inquiry point and friction between languages, ideas, practices, literary institutions, female authors, and female voices in today's markets. Hybrid literature written by first and second-generation migrants and displaced people is part of a huger concept of transnational literature, which breaks down with the idea of national identity and transiting towards a new conceptualization of hybridity in the literary production, also based on the translation of writings to other languages. The research question is about the relation between the transnational literary works, written by migrant women, and the social change. Based on the conceptualization of the Bhabha's 'third space', I will analyse the relation between the positioning of female migrant writers of 21st century and the role of hybridity. The preliminary findings show, firstly, the idea of reconceptualising it appears in light of the complexity of migrant people's realities and sex-gender differences. By adopting an intersectional lens, focused on the dialectic between gender and race/ethnicity and class, this paper analyses the tensions embedded in the re-positioning of four female migrant writers and their transnational experience (self)reflected in their writings. The present research contributes to the scientific knowledge in the field of cross-cultural literary studies, crossed with the migration study, through questioning the changing gender role and relations in transnational migrant literature. In addition, the findings show that today's reflection on 'third space' theory in the diasporic literature seems an idea to be refined when migrant women are involved.
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Nowadays, RNA is an attractive target for the design of new small molecules with different pharmacological activities. Among several RNA molecules, long noncoding RNAs (lncRNAs) are extensively reported to be involved in cancer pathogenesis. In particular, the overexpression of lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) plays an important role in the development of multiple myeloma (MM). Starting from the crystallographic structure of the triple-helical stability element at the 3'-end of MALAT1, we performed a structure-based virtual screening of a large commercial database, previously filtered according to the drug-like properties. After a thermodynamic analysis, we selected five compounds for the in vitro assays. Compound M5, characterized by a diazaindene scaffold, emerged as the most promising molecule enabling the destabilization of the MALAT1 triplex structure and antiproliferative activity on in vitro models of MM. M5 is proposed as a lead compound to be further optimized for improving its affinity toward MALAT1.
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RNA Longo não Codificante , RNA Longo não Codificante/genética , RNA Longo não Codificante/química , Relação Estrutura-AtividadeRESUMO
BACKGROUND: We developed a 13-mer locked nucleic acid (LNA) inhibitor of miR-221 (LNA-i-miR-221) with a full phosphorothioate (PS)-modified backbone. This agent downregulated miR-221, demonstrated anti-tumor activity against human xenografts in mice, and favorable toxicokinetics in rats and monkeys. Allometric interspecies scaling allowed us to define the first-in-class LNA-i-miR-221 safe starting dose for the clinical translation. METHODS: In this first-in-human, open-label, dose-escalation phase 1 trial, we enrolled progressive cancer patients (aged ≥ 18 years) with ECOG 0-2 into 5 cohorts. The treatment cycle was based on a 30-min IV infusion of LNA-i-miR-221 on 4 consecutive days. Three patients within the first cohort were treated with 2 cycles (8 infusions), while 14 patients were treated with a single course (4 infusions); all patients were evaluated for phase 1 primary endpoint. The study was approved by the Ethics Committee and Regulatory Authorities (EudraCT 2017-002615-33). RESULTS: Seventeen patients received the investigational treatment, and 16 were evaluable for response. LNA-i-miR-221 was well tolerated, with no grade 3-4 toxicity, and the MTD was not reached. We recorded stable disease (SD) in 8 (50.0%) patients and partial response (PR) in 1 (6.3%) colorectal cancer case (total SD + PR: 56.3%). Pharmacokinetics indicated non-linear drug concentration increase across the dose range. Pharmacodynamics demonstrated concentration-dependent downregulation of miR-221 and upregulation of its CDKN1B/p27 and PTEN canonical targets. Five mg/kg was defined as the recommended phase II dose. CONCLUSIONS: The excellent safety profile, the promising bio-modulator, and the anti-tumor activity offer the rationale for further clinical investigation of LNA-i-miR-221 (ClinTrials.Gov: NCT04811898).
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MicroRNAs , Neoplasias , Humanos , MicroRNAs/genética , MicroRNAs/uso terapêutico , Neoplasias/tratamento farmacológico , Oligonucleotídeos/uso terapêuticoRESUMO
Sulfur is an essential element of life. Thiol-containing metabolites in all organisms are involved in the regulation of diverse biological processes. Especially, the microbiome produces bioactive metabolites or biological intermediates of this compound class. The analysis of thiol-containing metabolites is challenging due to the lack of specific tools, making these compounds difficult to investigate selectively. We have now developed a new methodology comprising bicyclobutane for chemoselective and irreversible capturing of this metabolite class. We utilized this new chemical biology tool immobilized onto magnetic beads for the investigation of human plasma, fecal samples, and bacterial cultures. Our mass spectrometric investigation detected a broad range of human, dietary and bacterial thiol-containing metabolites and we even captured the reactive sulfur species cysteine persulfide in both fecal and bacterial samples. The described comprehensive methodology represents a new mass spectrometric strategy for the discovery of bioactive thiol-containing metabolites in humans and the microbiome.
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BACKGROUND: Pronectins™ are a new class of fibronectin-3-domain 14th-derived (14Fn3) antibody mimics that can be engineered as bispecific T cell engager (BTCE) to redirect immune effector cells against cancer. We describe here the in vitro and in vivo activity of a Pronectin™ AXL-targeted first-in-class bispecific T cell engager (pAXLxCD3ε) against Epithelial Ovarian Cancer (EOC). METHODS: pAXLxCD3ε T-cell mediated cytotoxicity was evaluated by flow cytometry and bioluminescence. pAXLxCD3ε mediated T-cell infiltration, activation and proliferation were assessed by immunofluorescence microscopy and by flow cytometry. Activity of pAXLxCD3ε was also investigated in combination with poly-ADP ribose polymerase inhibitors (PARPi). In vivo antitumor activity of pAXLxCD3ε was evaluated in immunocompromised (NSG) mice bearing intraperitoneal or subcutaneous EOC xenografts and immunologically reconstituted with human peripheral blood mononuclear cells (PBMC). RESULTS: pAXLxCD3ε induced dose-dependent cytotoxicity by activation of T lymphocytes against EOC cells, regardless of their histologic origin. The addition of PARPi to cell cultures enhanced pAXLxCD3ε cytotoxicity. Importantly, in vivo, pAXLxCD3ε was highly effective against EOC xenografts in two different NSG mouse models, by inhibiting the growth of tumor cells in ascites and subcutaneous xenografts. This effect translated into a significantly prolonged survival of treated animals. CONCLUSION: pAXLxCD3ε is an active therapeutics against EOC cells providing a rational for its development as a novel agent in this still incurable disease. The preclinical validation of a first-in-class agent opens the way to the development of a new 14Fn3-based scaffold platform for the generation of innovative immune therapeutics against cancer.
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Anticorpos Biespecíficos , Neoplasias Ovarianas , Humanos , Camundongos , Animais , Feminino , Leucócitos Mononucleares , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Linfócitos T , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Complexo CD3RESUMO
Bacterial host cell invasion has routinely been investigated by gentamicin protection assays, which are laborsome and suffer from pronounced experimental noise. This chapter describes an internally controlled, medium- to high-throughput method that resolves the capacity of multiple Salmonella virulence factor mutant strains to bind and invade host cells. The method, widely applicable to also other pathogens, is based on the combination of consortia of genetically tagged isogenic bacterial strains and a modified gentamicin protection assay. These protocols provide a flexible tool box to stringently quantify host cell binding and invasive properties of different mutants. Moreover, the method can be applied to both infections of cultured host cells and in vivo animal models, providing a comparable genetic readout, which greatly facilitates comparisons across experimental models.