Membranous urethral length is the single independent predictor of urinary continence recovery at 12 months following Retzius-sparing robot-assisted radical prostatectomy.

The influence of anatomical parameters on urinary continence (UC) after Retzius-sparing robot-assisted radical prostatectomy (RS-RARP) remains uncharted. Our objective was to evaluate their association with UC at 3, 6 and 12 months post-operatively. Data from patients who underwent RS-RARP were prospectively collected. Continence was defined as no pad use. Anatomic variables were measured on preoperative magnetic resonance imaging (MRI). Regression analyses were performed to identify predictors of UC at each time point. We included 158 patients with a median age of 60 years, most of whom had a localized tumor (≤ cT2). On multivariate analyses, at 3 months post-surgery, urinary incontinence (UI) rises with age, odds ratio (OR) 1.07 [95% confidence interval (CI) 1.004-1.142] and with prostate volume (PV), OR 1.029 (95% CI 1.006-1.052); it reduces with longer membranous urethral length (MUL), OR 0.875 (95% CI 0.780-0.983) and with higher membranous urethral volume (MUV), OR 0.299 (95% CI 0.121-0.737). At 6 months, UI rises with PV, OR 1.033 (95% CI 1.011-1.056) and decreases with MUV, OR 0.1504 (95% CI 0.050-0.444). Significantly, at 12 months post-surgery, the only predictor of UI is MUL, OR 0.830 (95% CI 0.706-0.975), establishing a threshold associated with a risk of UI of 5% (MUL > 15 mm) in opposition to a risk of 25% (MUL < 10 mm). This single institutional study requires external validation. To our knowledge, this is the first prospective cohort study supporting MUL as the single independent predictor of UC at 12 months post-surgery. By establishing MUL thresholds, we enable precise patient counseling.

A Case Study of a Rare Undifferentiated Spindle Cell Sarcoma of the Penis: Establishment and Characterization of Patient-Derived Models.

Rare sarcomas present significant treatment challenges compared to more prevalent soft tissue sarcomas due to limited treatment options and a poor understanding of their biology. This study investigates a unique case of penile sarcoma, providing a comprehensive morphological and molecular analysis. Through the creation of experimental patient-derived models-including patient-derived xenograft (PDX), 3D, and monolayer primary cultures-we successfully replicated crucial molecular traits observed in the patient's tumor, such as smooth muscle actin and CD99 expression, along with specific mutations in genes like TSC2 and FGFR4. These models are helpful in assessing the potential for an in-depth exploration of this tumor's biology. This comprehensive approach holds promise in identifying potential therapeutic avenues for managing this exceedingly rare soft tissue sarcoma.

Genetically modified ZIKA virus as a microRNA-sensitive oncolytic virus against central nervous system tumors.

Here we introduce a first-in-class microRNA-sensitive oncolytic Zika virus (ZIKV) for virotherapy application against central nervous system (CNS) tumors. The described methodology produced two synthetic modified ZIKV strains that are safe in normal cells, including neural stem cells, while preserving brain tropism and oncolytic effects in tumor cells. The microRNA-sensitive ZIKV introduces genetic modifications in two different virus sites: first, in the established 3'UTR region, and secondly, in the ZIKV protein coding sequence, demonstrating for the first time that the miRNA inhibition systems can be functional outside the UTR RNA sites. The total tumor remission in mice bearing human CNS tumors, including metastatic tumor growth, after intraventricular and systemic modified ZIKV administration, confirms the promise of this virotherapy as a novel agent against brain tumors-highly deadly diseases in urgent need of effective advanced therapies.

Establishment and Comprehensive Molecular Characterization of an Immortalized Glioblastoma Cell Line from a Brazilian Patient.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults, with few effective treatment strategies. The research on the development of new treatments is often constrained by the limitations of preclinical models, which fail to accurately replicate the disease's essential characteristics. Herein, we describe the obtention, molecular, and functional characterization of the GBM33 cell line. This cell line belongs to the GBM class according to the World Health Organization 2021 Classification of Central Nervous System Tumors, identified by methylation profiling. GBM33 expresses the astrocytic marker GFAP, as well as markers of neuronal origin commonly expressed in GBM cells, such as ßIII-tubulin and neurofilament. Functional assays demonstrated an increased growth rate when compared to the U87 commercial cell line and a similar sensitivity to temozolamide. GBM33 cells retained response to serum starvation, with reduced growth and diminished activation of the Akt signaling pathway. Unlike LN-18 and LN-229 commercial cell lines, GBM33 is able to produce primary cilia upon serum starvation. In summary, the successful establishment and comprehensive characterization of this GBM cell line provide researchers with invaluable tools for studying GBM biology, identifying novel therapeutic targets, and evaluating the efficacy of potential treatments.

A novel program of infiltrative control in astrocytomas: ADAM23 depletion promotes cell invasion by activating γ-secretase complex.

Background: Infiltration is a life-threatening growth pattern in malignant astrocytomas and a significant cause of therapy resistance. It results in the tumor cell spreading deeply into the surrounding brain tissue, fostering tumor recurrence and making complete surgical resection impossible. We need to thoroughly understand the mechanisms underlying diffuse infiltration to develop effective therapies. Methods: We integrated in vitro and in vivo functional assays, RNA sequencing, clinical, and expression information from public data sets to investigate the role of ADAM23 expression coupling astrocytoma's growth and motility. Results: ADAM23 downregulation resulted in increased infiltration, reduced tumor growth, and improved overall survival in astrocytomas. Additionally, we show that ADAM23 deficiency induces γ-secretase (GS) complex activity, contributing to the production and deposition of the Amyloid-ß and release of NICD. Finally, GS ablation in ADAM23-low astrocytomas induced a significant inhibitory effect on the invasive programs. Conclusions: Our findings reveal a role for ADAM23 in regulating the balance between cell proliferation and invasiveness in astrocytoma cells, proposing GS inhibition as a therapeutic option in ADAM23 low-expressing astrocytomas.

Urinary continence recovery after Retzius-sparing robot-assisted radical prostatectomy in relation to surgeon experience.

Urinary incontinence is one of the main concerns for patients after radical prostatectomy. Differences in surgical experience among surgeons could partly explain the wide range of frequencies observed. Our aim was to evaluate the association between the surgeons` experience and center caseload with relation to urinary continence recovery after Retzius-sparing robot-assisted radical prostatectomy (RS-RARP). Prospective observational single-center study. Five surgeons consecutively operated 405 patients between July 2017 and February 2022. Continence recovery was evaluated with pad count and by employing the short form of the International Consultation on Incontinence Questionnaire (ICIQ-SF), pre- and postoperatively at 1 year. Non-parametric tests were used. Median age was 63 years, 30% of patients presented with local advanced disease; the positive surgical margin rate (over 3 mm length) was 16%. Complication rate was 1% (Clavien-Dindo > II). One year after surgery, continence was assessed in 282 patients, of whom 87% were pad free and 51% never leaked (ICIQ-SF = 0). With respect to the mean annual number of procedures per surgeon, divided in < 20, 20-39 and ≥ 40, pad-free rates were achieved in 93%, 85%, and 84% and absence of urine leak rates in 47%, 62% and 48% of patients, respectively. Postoperative median ICIQ-SF was five. We acknowledge the limitation of a 12-month follow-up and the fact that we are a medium-volume center. There is no statistically significant association between continence recovery, surgeon's experience and center caseload. Continence recovery at 1 year after surgery is adequate and robust to surgeon's experience.

Sub­chronic administration of lead alters markers of oxidative stress, acetylcholinesterase and Na+K+­ATPase activities in rat brain.

This study investigated the effects of sub­chronic administration of lead (Pb) acetate on thiobarbituric acid reactive substances (TBA­RS), total sulfhydryl content, protein carbonyl content, antioxidant enzymes (superoxide dismutase [SOD], catalase [CAT], glutathione peroxidase [GSH­Px]), acetylcholinesterase (AChE), and Na+K+­ATPase in the cerebral structures of rats. Male Wistar rats aged 60 days were treated with saline (control group) or Pb (treatment group), at various doses, by gavage, once a day for 35 days. The animals were sacrificed twelve hours after the last administration, and the cerebellum, hippocampus and cerebral cortex were removed. The results showed that Pb did not alter the evaluated oxidative stress parameters. Furthermore, Pb (64 and/or 128 mg/kg) altered SOD in the cerebellum, cerebral cortex and hippocampus. Pb (128 mg/kg) altered CAT in the cerebellum and cerebral cortex and GSH­Px in the cerebral cortex. Also, Pb (64 mg/kg and 128 mg/kg) altered GSH­Px in the cerebellum. Moreover, Pb (128 mg/kg) increased AChE in the hippocampus and decreased Na+K+­ATPase in the cerebellum and hippocampus. In conclusion, sub­chronic exposure to Pb (occupational and environmental intoxication) altered antioxidant enzymes, AChE, and Na+K+­ATPase, contributing to cerebral dysfunction.

Homocysteine May Decrease Glucose Uptake and Alter the Akt/GSK3ß/GLUT1 Signaling Pathway in Hippocampal Slices: Neuroprotective Effects of Rivastigmine and Ibuprofen.

Homocysteine (Hcy) is a risk factor for neurodegenerative diseases, such as Alzheimer's Disease, and is related to cellular and tissue damage. In the present study, we verified the effect of Hcy on neurochemical parameters (redox homeostasis, neuronal excitability, glucose, and lactate levels) and the Serine/Threonine kinase B (Akt), Glucose synthase kinase-3ß (GSK3ß) and Glucose transporter 1 (GLUT1) signaling pathway in hippocampal slices, as well as the neuroprotective effects of ibuprofen and rivastigmine alone or in combination in such effects. Male Wistar rats (90 days old) were euthanized and the brains were dissected. The hippocampus slices were pre-treated for 30 min [saline medium or Hcy (30 µM)], then the other treatments were added to the medium for another 30 min [ibuprofen, rivastigmine, or ibuprofen + rivastigmine]. The dichlorofluorescein formed, nitrite and Na+, K+-ATPase activity was increased by Hcy at 30 µM. Ibuprofen reduced dichlorofluorescein formation and attenuated the effect of Hcy. The reduced glutathione content was reduced by Hcy. Treatments with ibuprofen and Hcy + ibuprofen increased reduced glutathione. Hcy at 30 µM caused a decrease in hippocampal glucose uptake and GLUT1 expression, and an increase in Glial Fibrillary Acidic Protein-protein expression. Phosphorylated GSK3ß and Akt levels were reduced by Hcy (30 µM) and co-treatment with Hcy + rivastigmine + ibuprofen reversed these effects. Hcy toxicity on glucose metabolism can promote neurological damage. The combination of treatment with rivastigmine + ibuprofen attenuated such effects, probably by regulating the Akt/GSK3ß/GLUT1 signaling pathway. Reversal of Hcy cellular damage by these compounds may be a potential neuroprotective strategy for brain damage.

Structural Perspective into the Interaction of an Oncogenesis-Relevant pre-miRNA G-Quadruplex Ligand Carrier with the Protein Nucleolin.

The structural determinants of the interaction of the G-quadruplex (G4) motif found in precursor miRNA 149 (rG4) with the acridine orange derivative C8 , a G4 ligand stabilizer possessing anticancer activity, and the protein nucleolin (overexpressed in cancer cells) were investigated by Nuclear Magnetic Resonance (NMR) spectroscopy. For the rG4/C8 complex, the results revealed a strong stabilizing interaction between the aromatic core and the iodinated ring of the C8 ligand with the rG4 structure. The NMR study revealed also different interaction patterns between nucleolin and rG4 and nucleolin and rG4/C8 complex. In the absence of the ligand, rG4 establishes interactions with polar residues of the protein while for the rG4/C8 complex, these contacts are mainly established with amino acids that have hydrophobic side chains. However, nucleolin chemical shift perturbation studies in the presence of rG4 or rG4/C8 reveal the same location between domains 1 and 2 of the protein, which suggests that the rG4 and rG4/C8 complex bind in this region. This puzzling structural study opens a new framework to study rG4/ligand/nucleolin complexes that might impact the biogenesis of miRNA 149.

Evaluation of Usage of a Fracture Risk Assessment by FRAX Tool in Adults With Type 2 Diabetes Mellitus.

BACKGROUND: Fragility fractures are increasingly recognized as a complication of type 2 diabetes mellitus (T2DM). The FRAX-Port® is a calculation tool that assesses the 10-year risk of either major and hip fracture, integrating several clinical risk factors, including T2DM. We aimed to evaluate the fracture risk in adults with T2DM and determine the rate of patients at high risk for fracture under anti-osteoporotic therapy. METHODS: We developed a cross-sectional study, including a convenience sample of adults with T2DM, followed in our tertiary center between 2019 and 2022. Fracture risk was evaluated according to FRAX-Port®. RESULTS: One hundred adults were included, 54% male, with a mean age of 68.4±9.2 years. Respecting fracture risk factors, 17% had a previous fragility fracture, 12% had a history of hip fracture in their parents, 9% had active alcohol consumption, and 4% had active smoking. Additionally, 17% presented secondary osteoporosis, being the most frequent cause of systemic corticosteroid exposure (10%). Regarding diabetes-specific risk factors, 94% had a diabetes duration longer than five years; HbA1c greater than 7% in 70%; 42% had diabetic retinopathy, 33% had diabetic chronic kidney disease, 18% had peripheral neuropathy, and 7% had autonomic neuropathy; 83% were on insulin, 2% on canagliflozin and 1% on pioglitazone. According to the FRAX-Port®, the median probability of major fracture was 6.8% (IQR 6.9), and hip fracture was 2.4% (IQR 3.9). Fracture risk was high, intermediate, and low at 41%, 15%, and 44%, respectively. Lastly, 56% of participants should undergo bone densitometry and 45% had a formal recommendation to begin an anti-osteoporotic treatment. However, only 6% were under anti-osteoporotic therapy: bisphosphonates (5%) and denosumab (1%). CONCLUSIONS: More than a third of T2DM patients evaluated had a high fracture risk. We found that FRAX-Port® is an easy-to-apply tool, which helps in the decision to perform densitometry or to institute anti-osteoporotic therapy. Given the increasing prevalence of T2DM and the associated risk of falls, this study highlights the need to recognize the fracture risk in these patients, usually a forgotten complication during the screening of risk factors for adverse events in adults with T2DM.

Retzius-sparing robot-assisted radical prostatectomy in a medium size oncological center holds adequate oncological and functional outcomes.

Retzius-sparing robot-assisted radical prostatectomy (RS-RARP) has emerged as a surgical option for patients with prostatic cancer in high-volume centers. The objective is to assess oncological and functional outcomes when implementing RS-RARP in a medium-volume center without previous experience of robotic surgery. This is a prospective observational single-center study. Patients operated between July 2017 and April 2020 were divided into two consecutive groups, A and B, each with 104 patients. The surgeons had prior experience in laparoscopic surgery and underwent robotic training. Positive surgical margin (PSM) status, urinary continence, and erectile function projected by Kaplan-Meier curves, together with patient reported quality of life outcomes at 12 months post-surgery were documented. Median patient age was 63 years (IQR = 59-67), overall PSM rate were 33%, 28% for pT2 disease. Pre-operative values showed no significant difference between both groups. The rate of urinary continence dropped from 81 to 78% (SE = 5.7) (Group A) and from 90 to 72% (SE = 6.3) (Group B) using the International Consultation on Incontinence Questionnaire-Short Form. Baseline sexual function was regained in 41% (Group A) and 47% (Group B) of patients. The median Expanded Prostate Index Composite-26 total score decreased from 86 to 82. These outcomes relate favorably to prior reports. There was a clinically significant decrease in median operative time in the successive groups with post-operative complications occurring in less than 2% of surgical procedures overall. A 12-month follow-up suggests that RS-RARP may be safely introduced in a medium-volume center without previous experience of robotic surgery.

Changes in functional mobility of patients with solid tumors after discharge from intensive care unit / Cambios en la movilidad funcional de pacientes con tumores sólidos tras el alta de la unidad de cuidados intensivos / Mudanças na mobilidade funcional de pacientes com tumores sólidos após a alta da unidade de terapia intensiva

ABSTRACT This study aimed to analyze changes in the level of functional mobility (FM) between patients with solid tumors discharged from intensive care units (ICU) and hospital discharge and the possible factors associated with FM recovery. This is a retrospective cohort study based on the analysis of medical records of patients with solid tumors who were discharged from an oncology ICU from January 1, 2018 to February 28, 2020. The primary outcome was the change in FM after ICU discharge, considering the difference between the final score at ICU discharge and the final score at hospital discharge, estimated by the ICU Mobility Scale (IMS). The association between continuous variables and outcomes was performed by univariate linear regression analysis. In total, 65 patients with a median age of 61.4 years (interquartile range - IQR 54-69) were included. The mean length of hospital stay after discharge from the ICU was 19.0 days (±24.04). The mean IMS score at ICU discharge was 2.62 (±2.56) and the mean IMS score at hospital discharge was 6.08 (±3.26). Patients who underwent surgery to treat the primary tumor had a score 1.89 higher compared to those who did not undergo surgery (p=0.048). Therefore, we observed improvement in FM in patients with solid tumors between ICU discharge and hospital discharge, and patients who underwent surgery showed better FM.

RESUMEN Este estudio tuvo como objetivo analizar los cambios en la movilidad funcional (MF) de pacientes con tumores sólidos entre el alta de la unidad de cuidados intensivos (UCI) y el alta del hospital, y los posibles factores asociados con la recuperación de la MF. Se trata de un estudio de cohorte retrospectivo realizado desde el análisis de historias clínicas de pacientes con tumores sólidos que fueron dados de alta de la UCI oncológica entre el 1 de enero de 2018 y el 28 de febrero de 2020. El resultado primario fue el cambio en la MF después del alta de la UCI considerando la diferencia entre la puntuación final al alta de la UCI y la puntuación final al alta del hospital, que se calculó mediante la ICU mobility scale (IMS). La asociación entre las variables continuas y los resultados se realizó mediante análisis de regresión lineal univariante. Se incluyeron un total de 65 pacientes con mediana de edad de 61,4 años (rango intercuartílico -RIC 54-69). La estancia media de hospitalización tras el alta de la UCI fue de 19,0 días (±24,04). La puntuación media de IMS al alta de la UCI fue de 2,62 (±2,56), y la del alta del hospital 6,08 (±3,26). Los pacientes que se sometieron a cirugía para tratar el tumor primario tuvieron una puntuación 1,89 veces mayor en comparación con los que no se sometieron a tratamiento quirúrgico (p=0,048). Se concluye que hubo una mejoría en la MF en pacientes con tumores sólidos entre el alta de la UCI y el alta del hospital, y los pacientes sometidos a cirugía mostraron una mejor recuperación de la MF.

RESUMO Este estudo teve como objetivo analisar as mudanças na mobilidade funcional (MF) de pacientes com tumores sólidos entre a alta da unidade de terapia intensiva (UTI) e a alta hospitalar e os possíveis fatores associados à recuperação da MF. Trata-se de um estudo de coorte retrospectivo baseado na análise de prontuários de pacientes com tumores sólidos que receberam alta da UTI de uma unidade oncológica entre 1º de janeiro de 2018 e 28 de fevereiro de 2020. O desfecho primário foi a mudança na MF após a alta da UTI considerando a diferença entre a pontuação final na alta da UTI e a pontuação final na alta hospitalar, calculada através da ICU mobility scale (IMS). A associação entre as variáveis contínuas e os desfechos foi realizada por meio da análise de regressão linear univariada. No total, foram incluídos 65 pacientes com idade mediana de 61,4 anos (variação interquartil - IQR 54-69). O tempo médio de internação após a alta da UTI foi de 19,0 dias (±24,04). A pontuação média da IMS no momento da alta da UTI foi de 2,62 (±2,56), e a pontuação média da IMS no momento da alta hospitalar foi de 6,08 (±3,26). Os pacientes que realizaram cirurgia para o tratamento do tumor primário tiveram uma pontuação 1,89 vez maior em comparação aos que não foram submetidos a tratamento cirúrgico (p=0,048). Concluindo, foi observada melhora da MF em pacientes com tumores sólidos entre a alta da UTI e a alta hospitalar, e os pacientes submetidos à cirurgia apresentaram uma melhor recuperação da MF.

Whipple's disease - A typical endoscopic finding of a rare disease.

A 49-year-old female presented with a 5-month course of diarrhoea, nocturn abdominal pain, asthenia, and weight loss of 30% of her body mass in three months. The patient had also a four-year medical history of bilateral mechanic gonalgy and arthralgias of the metacarpophalangeal and interphalangeal joints, despite treatment with prednisolone. On examination the patient had hyperpigmentation of the face and thorax, low-grade fever, and a BMI of 15,8 Kg/m2. Diarrhoea was documented with watery stools seven times per day despite loperamide, brownish, with no visible blood or mucous. Since the upper GI endoscopy and colonoscopy had no macroscopic abnormalities, the patient underwent a capsule endoscopy, which revealed continuous mucosal lesion with lymphangiectasia, oedema, villous atrophy and areas of denudation with hematinic punctate from the duodenum to the ileum. Diagnosis of Whipple's Disease was made with typical histology findings in duodenum material and a positive PCR for Tropheryma whipplei.

Levothyroxine malabsorption or pseudomalabsorption? A question in the management of refractory hypothyroidism.

Purpose: The levothyroxine absorption test (LT4AT) is an important tool for distinguishing hypothyroidism due to malabsorption from 'pseudomalabsorption' conditions. Our aim was to review our institution's LT4AT results and assess its role in the management of patients with refractory hypothyroidism. Methods: We performed a retrospective study of all patients evaluated for refractory hypothyroidism who underwent LT4AT in our tertiary center between 2014 and 2020. Its results and the impact on thyroid function management during follow-up were assessed. Results: Ten female patients were included with a mean age of 40 years (min-max: 26-62). Mean weight was 72 kg (min-max: 43-88) and baseline LT4 dosage ranged from 2.5 to 5.3 µg/kg/day. The most common causes of hypothyroidism were postsurgical in 50% (n = 5) and autoimmune in 20% (n = 2). During LT4AT, normal LT4 absorption was found in all but one individual (mean FT4 increase of 231%, min-max: 85-668). The only patient with objective LT4 absorption impairment (maximal increase of 48% by hour 5) presented also Helicobacter pylori gastritis and prior history of 'intestinal surgery' during childhood. No adverse events were reported during any of the LT4ATs. During follow-up (median 11.5 months (IQR 23)), three patients obtained euthyroidism and six had improved their hypothyroidism state. Conclusions: The LT4AT is an effective and safe way to assess refractory hypothyroidism and provides valuable information to distinguish LT4 malabsorption from 'pseudomalabsorption'. Our data suggest that most patients with suspicious LT4 malabsorption perform normally during LT4AT. This test provides relevant information for better management of patients with refractory hypothyroidism.

G-Quadruplex Aptamer-Ligand Characterization.

In this work we explore the structure of a G-rich DNA aptamer termed AT11-L2 (TGGTGGTGGTTGTTGTTGGTGGTGGTGGT; derivative of AT11) by evaluating the formation and stability of G-quadruplex (G4) conformation under different experimental conditions such as KCl concentration, temperature, and upon binding with a variety of G4 ligands (360A, BRACO-19, PDS, PhenDC3, TMPyP4). We also determined whether nucleolin (NCL) can be a target of AT11-L2 G4. Firstly, we assessed by circular dichroism, UV and NMR spectroscopies the formation of G4 by AT11-L2. We observed that, for KCl concentrations of 65 mM or less, AT11-L2 adopts hybrid or multiple topologies. In contrast, a parallel topology predominates for buffer containing 100 mM of KCl. The Tm of AT11-L2 in 100 mM of KCl is 38.9 °C, proving the weak stability of this sequence. We also found that upon titration with two molar equivalents of 360A, BRACO-19 and PhenDC3, the G4 is strongly stabilized and its topology is maintained, while the addition of 3.5 molar equivalents of TMPyP4 promotes the disruption of G4. The KD values between AT11-L2 G4, ligands and NCL were obtained by fluorescence titrations and are in the range of µM for ligand complexes and nM when adding NCL. In silico studies suggest that four ligands bind to the AT11-L2 G4 structure by stacking interactions, while the RBD1,2 domains of NCL interact preferentially with the thymines of AT11-L2 G4. Finally, AT11-L2 G4 co-localized with NCL in NCL-positive tongue squamous cell carcinoma cell line.

Biological activities and physicochemical characterization of alkaline lignins obtained from branches and leaves of Buchenavia viridiflora with potential pharmaceutical and biomedical applications.

In this work, we investigated in vitro different biological activities of alkaline lignins extracted from the species Buchenavia viridiflora, a tree from the Amazon rainforest used as a wood product. The chemical composition results for the twig and leaves were, respectively (%): cellulose (30.88 and 24. 28), hemicellulose (21.62 and 23.03), lignin (29.93 and 25.46), extractives (13.06 and 20.52), and ash (4.51 and 6.72). The yield was higher for the lignin of the branches (67.9 %) when compared to the leaves (60.2 %). Lignins are of the GSH type, low molecular weight and thermally stable. They promoted moderate to low antioxidant activity, highlighting the lignin of the branches, which presented an IC50 of 884.56 µg/mL for the DPPH assay and an IC50 of 14.08 µg/mL for ABTS. In the cytotoxicity assays, they showed low toxicity against macrophage cells (IC50 28.47 and 22.58 µg/mL). In addition, they were not cytotoxic against splenocytes and erythrocytes at concentrations ranging from 100 to 6.25 µg/mL. These were able to promote splenocyte proliferation and induce the production of anti-inflammatory cytokines. And inhibit the growth of tumor cells with IC50 ranging from 12.63 to values >100 µg/mL and microbial at a concentration of 512 µg/mL. Finally, they showed antiparasitic activity by inhibiting the growth of chloroquine-sensitive and resistant Plasmodium falciparum strains. These findings reinforce that the lignins in this study are promising for potential pharmaceutical and biomedical applications.

Immune Checkpoint Blockade via PD-L1 Potentiates More CD28-Based than 4-1BB-Based Anti-Carbonic Anhydrase IX Chimeric Antigen Receptor T Cells.

The complete regression of clear cell renal cell carcinoma (ccRCC) obtained pre-clinically with anti-carbonic anhydrase IX (CAIX) G36 chimeric antigen receptor (CAR) T cells in doses equivalent to ≅108 CAR T cells/kg renewed the potential of this target to treat ccRCC and other tumors in hypoxia. The immune checkpoint blockade (ICB) brought durable clinical responses in advanced ccRCC and other tumors. Here, we tested CD8α/4-1BB compared to CD28-based anti-CAIX CAR peripheral blood mononuclear cells (PBMCs) releasing anti-programmed cell death ligand-1 (PD-L1) IgG4 for human ccRCC treatment in vitro and in an orthotopic NSG mice model in vivo. Using a ≅107 CAR PBMCs cells/kg dose, anti-CAIX CD28 CAR T cells releasing anti-PD-L1 IgG highly decrease both tumor volume and weight in vivo, avoiding the occurrence of metastasis. This antitumoral superiority of CD28-based CAR PBMCs cells compared to 4-1BB occurred under ICB via PD-L1. Furthermore, the T cell exhaustion status in peripheral CD4 T cells, additionally to CD8, was critical for CAR T cells efficiency. The lack of hepatotoxicity and nephrotoxicity upon the administration of a 107 CAR PMBCs cells/kg dose is the basis for carrying out clinical trials using anti-CAIX CD28 CAR PBMCs cells releasing anti-PD-L1 antibodies or anti-CAIX 4-1BB CAR T cells, offering exciting new prospects for the treatment of refractory ccRCC and hypoxic tumors.

Interleukin-6 Signaling in Triple Negative Breast Cancer Cells Elicits the Annexin A1/Formyl Peptide Receptor 1 Axis and Affects the Tumor Microenvironment.

Annexin A1 (AnxA1) is a pleiotropic protein that exerts essential roles in breast cancer (BC) growth and aggressiveness. In our previous work, we described the autocrine signaling of AnxA1 through formyl peptide receptor 1 (FPR1) in the triple-negative (TN) BC cell line, MDA-MB-231. Here, we aimed to describe the interaction between the AnxA1/FPR1 and the Interleukin-6 (IL-6) signaling pathways and their role in the tumor microenvironment (TME). First, we demonstrated that AnxA1 and IL-6 expression levels are correlated in BC tissue samples. In three TNBC cell lines, overexpression of both AnxA1 and IL-6 was also identified. Next, we inhibited FPR1, the IL-6 receptor and STAT3 in both MDA-MB-231 and MDA-MB-157 cells. The FPR1 inhibition led to increased levels of IL-6 and secreted AnxA1 in both cell lines. On the other side, inhibition of the IL-6 receptor or STAT3 led to the impairment of AnxA1 secretion, suggesting the essential role of the IL-6 signaling cascade in the activation of the AnxA1/FPR1 autocrine axis. Finally, we described the interaction between IL-6 and the AnxA1/FPR1 pathways and their role on the TME by analyzing the effect of supernatants derived from MDA-MB-231 and MDA-MB-157 cells under the inhibition of FPR1 or IL-6 signaling on fibroblast cell motility.

Stabilization of a DNA aptamer by ligand binding.

G-rich aptamers such as AS1411 are small oligonucleotides that present several benefits comparatively to monoclonal antibodies, since they are easier to manufacture and store, have small size and do not stimulate an immune response. We analyzed AT11-B1, a modified sequence of AT11 (itself a modified version of AS1411), in which one thymine was removed from the bulge region. We studied G-quadruplex (G4) formation/stabilization using PhenDC3, PDS, BRACO-19, TMPyP4 and 360A ligands by different biophysical techniques, namely circular dichroism (CD), Förster resonance energy transfer (FRET-melting) and nuclear magnetic resonance (NMR). The CD spectra showed that AT11-B1 adopts a predominant G4 of parallel topology when the buffer contains KCl or when ligands are added. PhenDC3 induced a ΔTm of 30 °C or more of the G4 structure as shown by CD- and FRET-melting experiments. The ligands demonstrate high affinity for AT11-B1 G4 and the NMR studies revealed that the AT11-B1 G4 involves four G-tetrad layers. The in silico studies suggest that all ligands bind AT11-B1 G4, namely, by stacking interactions, with the possible exception of PDS that may bind to the loop/groove interface. In addition, molecular dynamics simulations revealed that nucleolin (NCL) interacts with the AT11-B1 G4 structure through the RNA binding domain (RBD) 2 and the 12-residue linker between RBD1,2. Moreover, AT11-B1 G4 was internalized into a NCL-positive tongue squamous cell carcinoma cell line. In a nutshell, this study may help the identification of the ligands scaffolds to bind and stabilize AT11-B1, improving the targeting towards NCL that is overexpressed in cancer cells.