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1.
Gynecol Oncol ; 179: 79-84, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37944329

RESUMO

Brachytherapy (BT) is an integral component of treatment for patients with locally advanced cervical cancer, significantly improving local control and overall survival. There is an overall trend of decreased utilization of BT in United States (US) in the last few decades with around 50% of patients being treated without BT. The cause of decreased utilization is multifactorial including physician comfort, facility volume, low reimbursements rates and costs of starting and maintaining a brachytherapy program. This decrease coincides with an increase in the use of newer advanced techniques like intensity modulated radiation therapy (IMRT) and stereotactic body radiation therapy (SBRT) boost resulting in inferior oncological outcomes and increased toxicity. Moreover, racial and socioeconomic disparities in BT utilization have been widely reported in the US. Various factors including age, race, socio-economic status, location, facility type, facility volume and insurance status result in limited access to brachytherapy, which jeopardizes oncologic outcomes. This comprehensive review discusses the BT utilization in the US, examines the impact of race and socioeconomic factors on BT utilization, and highlights its impact on outcomes.


Assuntos
Braquiterapia , Radioterapia de Intensidade Modulada , Neoplasias do Colo do Útero , Feminino , Humanos , Estados Unidos/epidemiologia , Neoplasias do Colo do Útero/radioterapia , Braquiterapia/métodos , Fatores Socioeconômicos , Radioterapia de Intensidade Modulada/métodos , Classe Social
2.
Environ Res ; 231(Pt 1): 116155, 2023 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-37196692

RESUMO

Climatic conditions have been shown as a major driver of the fate of Hg in forest ecosystems at a global scale, but less is known about climatic effects at shorter scales. This study assesses whether the concentration and pools of Hg in soils collected from seventeen Pinus pinaster stands describing a coastal-inland transect in SW Europe vary along a regional climatic gradient. In each stand, samples of the organic subhorizons (OL, OF + OH) and the mineral soil (up to 40 cm) were collected and some general physico-chemical properties and total Hg (THg) were analyzed. Total Hg was significantly higher in the OF + OH than in the OL subhorizons (98 and 38 µg kg-1, respectively), favored by a greater organic matter humification in the former. In the mineral soil, mean THg values decreased with depth, ranging from 96 µg kg-1 in the 0-5 cm layers to 54 µg kg-1 in the deepest layers (30-40 cm), respectively. The average Hg pool (PHg) was 0.30 mg m-2 in the organic horizons (92% accumulated in the OF + OH subhorizons), and 27.4 mg m-2 in the mineral soil. Changes in climatic factors, mainly precipitation, along the coast-inland transect resulted in a remarkable variation of THg in the OL subhorizons, consistent with their role as the first receiver of atmospheric Hg inputs. The high precipitation rate and the occurrence of fogs in coastal areas characterized by the oceanic influence would explain the higher THg found in the uppermost soil layers of pine stands located close to the coastline. The regional climate is key to the fate of mercury in forest ecosystems by influencing the plant growth and subsequent atmospheric Hg uptake, the atmospheric Hg transference to the soil surface (wet and dry deposition and litterfall) and the dynamics that determine net Hg accumulation in the forest floor.


Assuntos
Mercúrio , Pinus , Poluentes do Solo , Ecossistema , Solo/química , Monitoramento Ambiental , Mercúrio/análise , Florestas , Europa (Continente) , Poluentes do Solo/análise
3.
Am J Perinatol ; 2023 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-37339676

RESUMO

OBJECTIVE: Given the rising rates of maternal morbidity and mortality in the United States and the contribution of mental illness, especially among individuals living in underresourced communities, the objective was to evaluate the prevalence of unmet health-related social needs and their impact on perinatal mental health outcomes. STUDY DESIGN: This was a prospective observational study of postpartum patients residing within regions with increased rates of poor perinatal outcomes and sociodemographic disparities. Patients were enrolled in a multidisciplinary public health initiative "extending Maternal Care After Pregnancy (eMCAP)" between October 1, 2020 and October 31, 2021. Unmet health-related social needs were assessed at delivery. Symptoms of postpartum depression and anxiety were evaluated at 1 month postpartum utilizing the Edinburgh Postnatal Depression Scale (EPDS) and Generalized Anxiety Disorder-7 (GAD7) screening tools, respectively. Mean EPDS and GAD7 scores and odds of screening positive (scoring ≥ 10) were compared among individuals with and without unmet health-related social needs with p < 0.05 considered significant. RESULTS: Of participants enrolled in eMCAP, 603 completed at least one EPDS or GAD7 at 1 month. Most had at least one social need, most commonly dependence on social programs for food (n = 413/603; 68%). Individuals lacking transportation to medical (odds ratio [OR]: 4.0, 95% confidence interval [CI]: 1.2-13.32) and nonmedical appointments (OR: 4.17, 95% CI: 1.08-16.03) had significantly higher odds of screening positive on EPDS while participants lacking transportation to medical appointments (OR: 2.73, 95% CI: 0.97-7.70) had significantly higher odds of screening positive on GAD7. CONCLUSION: Among postpartum individuals in underserved communities, social needs correlate with higher depression and anxiety screening scores. This highlights the need to address social needs to improve maternal mental health. KEY POINTS: · Social needs are prevalent among underserved patients.. · Needs can be assessed in a structured or freeform manner.. · Unmet needs correlate with poor mental health outcomes.. · Similar needs correlate with depression and anxiety..

4.
Int J Obes (Lond) ; 46(3): 661-668, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34974543

RESUMO

BACKGROUND: Patients with obesity have an increased risk for adverse COVID-19 outcomes. Body mass index (BMI) does not acknowledge the health burden associated this disease. The performance of the Edmonton Obesity Staging System (EOSS), a clinical classification tool that assesses obesity-related comorbidity, is compared with BMI, with respect to adverse COVID-19 outcomes. METHODS: 1071 patients were evaluated in 11 COVID-19 hospitals in Mexico. Patients were classified into EOSS stages. Adjusted risk factors for COVID-19 outcomes were calculated and survival analysis for mechanical ventilation and death was carried out according to EOSS stage and BMI category. RESULTS: The risk for intubation was higher in patients with EOSS stages 2 and 4 (HR 1.42, 95% CI 1.02-1.97 and 2.78, 95% CI 1.83-4.24), and in patients with BMI classes II and III (HR 1.71, 95% CI 1.06-2.74, and 2.62, 95% CI 1.65-4.17). Mortality rates were significantly lower in patients with EOSS stages 0 and 1 (HR 0.62, 95% CI 0.42-0.92) and higher in patients with BMI class III (HR 1.58, 95% CI 1.03-2.42). In patients with a BMI ≥ 25 kg/m2, the risk for intubation increased with progressive EOSS stages. Only individuals in BMI class III showed an increased risk for intubation (HR 2.24, 95% CI 1.50-3.34). Mortality risk was increased in EOSS stages 2 and 4 compared to EOSS 0 and 1, and in patients with BMI class II and III, compared to patients with overweight. CONCLUSIONS: EOSS was associated with adverse COVID-19 outcomes, and it distinguished risks beyond BMI. Patients with overweight and obesity in EOSS stages 0 and 1 had a lower risk than patients with normal weight. BMI does not adequately reflect adipose tissue-associated disease, it is not ideal for guiding chronic-disease management.


Assuntos
COVID-19 , Obesidade , Adulto , Idoso , COVID-19/complicações , COVID-19/epidemiologia , COVID-19/mortalidade , Comorbidade , Feminino , Hospitalização , Humanos , Masculino , Pessoa de Meia-Idade , Obesidade/complicações , Obesidade/epidemiologia , Obesidade/fisiopatologia , Estudos Retrospectivos , Índice de Gravidade de Doença , Resultado do Tratamento
5.
Environ Res ; 215(Pt 1): 114223, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36063908

RESUMO

Vegetation and climate are critical in the biogeochemical cycle of Hg in forest ecosystems. The study assesses the influence of needle age and precipitation on the accumulation of Hg in needle biomass and its deposition by litterfall in thirty-one pine plantations spread throughout two biogeographical regions in SW Europe. Well-developed branches of Pinus pinaster were sampled and pine needles were classified according to 4 age classes (y0, y1, y2, y3). The concentration of total Hg (THg) was analyzed in the samples and Hg content in needle biomass and its deposition by litterfall were estimated. The concentration of total Hg (THg) increased with needle age ranging from 9.1 to 32.7 µg Hg kg-1 in the youngest and oldest needles, respectively. The rate of Hg uptake (HgR) three years after needle sprouting was 10.2 ± 2.3 µg Hg kg-1 yr-1, but it decreased with needle age probably due to a diminution in photosynthetic activity as needles get older. The average total Hg stored in needle biomass (HgWt) ranged from 5.6 to 87.8 mg Hg ha-1, with intermediate needle age classes (y1 and y2) accounting for 70% of the total Hg stored in the whole needle biomass. The average deposition flux of Hg through needle litterfall (HgLt) was 1.5 µg Hg m-2 yr-1, with the y2 and y3 needles contributing most to the total Hg flux. The spatial variation of THg, HgWt and HgLt decreased from coastal pine stands, characterized by an oceanic climate, to inland pine stands, a feature closely related to the dominant precipitation regime in the study area. Climatic conditions and needle age are the main factors affecting Hg accumulation in tree foliage, and should be considered for an accurate assessment of forest Hg pools at a regional scale and their potential consequences in the functioning of terrestrial ecosystems.


Assuntos
Mercúrio , Traqueófitas , Ecossistema , Monitoramento Ambiental , Florestas , Mercúrio/análise , Árvores
6.
J Exp Biol ; 224(7)2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33912953

RESUMO

Animals adjust resource acquisition throughout life to meet changing physiological demands of growth, reproduction, activity and somatic maintenance. Wing-polymorphic crickets invest in either dispersal or reproduction during early adulthood, providing a system in which to determine how variation in physiological demands, determined by sex and life history strategy, impact nutritional targets, plus the consequences of nutritionally imbalanced diets across life stages. We hypothesized that high demands of biosynthesis (especially oogenesis in females) drive elevated resource acquisition requirements and confer vulnerability to imbalanced diets. Nutrient targets and allocation into key tissues associated with life history investments were determined for juvenile and adult male and female field crickets (Gryllus lineaticeps) when given a choice between two calorically equivalent but nutritionally imbalanced (protein- or carbohydrate-biased) artificial diets, or when restricted to one imbalanced diet. Flight muscle synthesis drove elevated general caloric requirements for juveniles investing in dispersal, but flight muscle quality was robust to imbalanced diets. Testes synthesis was not costly, and life history investments by males were insensitive to diet composition. In contrast, costs of ovarian synthesis drove elevated caloric and protein requirements for adult females. When constrained to a carbohydrate-biased diet, ovary synthesis was reduced in reproductive morph females, eliminating their advantage in early life fecundity over the dispersal morph. Our findings demonstrate that nutrient acquisition modulates dispersal-reproduction trade-offs in an age- and sex-specific manner. Declines in food quality will thus disproportionately affect specific cohorts, potentially driving demographic shifts and altering patterns of life history evolution.


Assuntos
Gryllidae , Animais , Dieta , Ingestão de Alimentos , Feminino , Masculino , Reprodução , Asas de Animais
7.
Environ Res ; 193: 110552, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33271140

RESUMO

Mercury content of twelve podzols from NW Spain was studied to elucidate the main soil properties involved in the Hg accumulation of these soils. The highest average Hg concentrations (HgT) were found in the Bh and Bs horizons (64 and 105 µg kg-1), whereas the lowest occurred in the E horizons (15 µg kg-1). Moderate values of HgT were obtained for the A and C horizons (38 and 52 µg kg-1). The Hg enrichment factors revealed that the predominant origin of Hg in these soils is the atmosphere instead of the parent material. As it was shown by the PCA performed (which explained 82% of the variance of the data), the main soil characteristics involved in the pedogenetic processes of the studied podzols are organic matter and Al and Fe compounds. The stepwise linear regressions made described between 54% and 84% of the predicted Hg depending on the soil horizon. Besides a complex ensemble of biogeochemical reactions involved in the balance between input and outputs of Hg, the most influencing variable in the A horizons was organic C, moderate stability Al-humus complexes in the E horizons, Fe-humus complexes and pHw in the Bh horizons, Al-humus compounds in the Bs horizons and crystalline Al and Fe compounds in the C horizons. Therefore, Hg is mobilized from the A and E horizons bound to dissolved organic matter and precipitated in the illuvial horizons due to the saturation of the organic matter with metals. The immobilization of Hg in the subsuperficial horizons of podzols leads to different environmental benefits derived from the removal of Hg from the A horizons, more exposed to climate-induced and land use/cover changes that could potentially modify the dynamics of Hg in those superficial horizons.


Assuntos
Mercúrio , Poluentes do Solo , Monitoramento Ambiental , Florestas , Mercúrio/análise , Solo , Poluentes do Solo/análise , Espanha
8.
Semin Speech Lang ; 40(2): 124-137, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30795023

RESUMO

A growing body of research suggests that cases of speech sound errors that have not responded to previous intervention can sometimes be eliminated through speech therapy incorporating visual biofeedback. Aside from considerations related to the specific biofeedback type, acquisition and generalization of a motor plan may be linked to treatment intensity. Several researchers have raised the possibility that inadequate dosage levels may present a significant barrier to success. Thus, the current study aimed to assess the relationship between treatment intensity and treatment outcomes. Twenty-nine articles reporting the use of visual biofeedback intervention for speech sound disorder were identified and coded for treatment intensity using the cumulative intervention index and outcomes using mean level difference scores. Findings reveal small but significant relationships between measures of treatment intensity and efficacy, which should be interpreted with caution given the preliminary nature of this review. Further research in this area is necessary, as inconsistencies in reporting intensity and outcomes across studies underscore the need for more systematic terminology and reporting methods.


Assuntos
Biorretroalimentação Psicológica/métodos , Transtorno Fonológico/terapia , Fonoterapia/métodos , Percepção Visual , Criança , Humanos , Transtorno Fonológico/diagnóstico , Resultado do Tratamento
9.
Prev Med ; 109: 119-124, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29366818

RESUMO

The current study examined the nexus of neighborhood disorder-in the form of physical disorder (e.g., broken glass and vandalism) and social disorder (e.g., public drinking and lewd conduct)-and physical activity (PA) in urban public parks to inform public policy addressing chronic disease in at-risk populations. Five hundred and twenty-two unique observations were conducted in 22 public parks from March to September 2016. The study utilized the System for Observing Play and Recreation in Communities (SOPARC) to document age, gender, ethnicity, and PA level of park users. The Physical Activity Resource Assessment (PARA) was used to document observed physical and social disorder incivilities in public parks included in the current study. Males, adults, and Latina/os accounted for the largest number of park users, respectively. Significant PA differences were observed across gender, age, and ethnicity. Multiple linear regression controlling for gender, age, and ethnicity found physical disorder, but not social disorder, generally predicted PA reductions. While it has been demonstrated that physical disorder predicts PA reductions in low-income communities of color, this is the first study to reveal that physical disorder may lead to decreased PA in urban public parks. Thus, remediation of public park incivilities characterized by physical disorder, paired with community outreach, may lead to increased PA in at-risk communities.


Assuntos
Exercício Físico/fisiologia , Parques Recreativos/estatística & dados numéricos , Recreação , Determinantes Sociais da Saúde , Fatores Socioeconômicos , Adolescente , Adulto , Idoso , Criança , Pesquisa Participativa Baseada na Comunidade , Etnicidade/estatística & dados numéricos , Feminino , Humanos , Incivilidade , Masculino , Pessoa de Meia-Idade , Características de Residência , Comportamento Social
12.
J Surg Oncol ; 115(5): 615-618, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28168717

RESUMO

Cervical cancer is a preventable disease with a known etiology (human papillomavirus), effective preventive vaccines, excellent screening methods, and a treatable pre-invasive phase. Surgery is the primary treatment for pre-invasive and early-stage disease and can safely be performed in many low-resource settings. However, cervical cancer rates remain high in many areas of Latin America. This article presents a number of evidence-based strategies being implemented to improve cervical cancer outcomes in Latin America.


Assuntos
Neoplasias do Colo do Útero/terapia , Feminino , Acessibilidade aos Serviços de Saúde , Humanos , América Latina/epidemiologia , Programas de Rastreamento , Infecções por Papillomavirus/diagnóstico , Vacinas contra Papillomavirus/uso terapêutico , Prevenção Primária , Consulta Remota , Neoplasias do Colo do Útero/epidemiologia , Neoplasias do Colo do Útero/virologia
14.
Aquat Toxicol ; 268: 106862, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38359500

RESUMO

Weak, but environmentally relevant concentrations of contaminants can have subtle, yet important, impacts on organisms, which are often overlooked due to the lack of acute impacts and the timing of exposure. Thus, recognizing simple, non-invasive markers of contamination events is essential for early detection and addressing the effects of exposure to weak environmental contaminants. Here, we tested whether exposure to an environmentally relevant concentration of Bisphenol-A (BPA), a common and persistent contaminant in aquatic systems, affects the lateralization of adult zebrafish (Danio rerio), a widely used model organism in ecotoxicology. We found that 73.5% of adult zebrafish displayed a left-side bias when they approached a visual cue, but that those exposed to weak BPA (0.02 mg/L) for 7 days did not exhibit laterality. Only 47.1% displayed a left-side bias. We found no differences in activity level and visual sensitivity, motor and sensory mechanisms, that regulate lateralized responses and that were unaffected by weak BPA exposure. These findings indicate the reliability of laterality as a simple measure of contaminant exposure and for future studies of the detailed mechanisms underlying subtle and complex behavioral effects to pollutants.


Assuntos
Poluentes Químicos da Água , Peixe-Zebra , Animais , Reprodutibilidade dos Testes , Poluentes Químicos da Água/toxicidade , Fenóis/toxicidade , Compostos Benzidrílicos/toxicidade
15.
Sci Rep ; 13(1): 16398, 2023 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-37773260

RESUMO

Phenotypes that allow animals to detect, weather, and predict changes efficiently are essential for survival in fluctuating environments. Some phenotypes may remain specialized to suit an environment perfectly, while others become more plastic or generalized, shifting flexibly to match current context or adopting a form that can utilize a wide range of contexts. Here, we tested the differences in behavior, morphology, sensory and metabolic physiology between wild zebrafish (Danio rerio) in highly variable fast-flowing rivers and still-water sites. We found that river zebrafish moved at higher velocities than did still-water fish, had lower oxygen demands, and responded less vigorously to small changes in flow rate, as we might expect for fish that are well-suited to high-flow environments. River zebrafish also had less streamlined bodies and were more behaviorally plastic than were still-water zebrafish, both features that may make them better-suited to a transitional lifestyle. Our results suggest that zebrafish use distinct sensory mechanisms and metabolic physiology to reduce energetic costs of living in fast-flowing water while relying on morphology and behavior to create flexible solutions to a challenging habitat. Insights on animals' reliance on traits with different outcomes provide a framework to better understand their survival in future environmental fluctuations.


Assuntos
Poluentes Químicos da Água , Peixe-Zebra , Animais , Peixe-Zebra/fisiologia , Rios , Ecossistema , Comportamento Animal , Agricultura , Poluentes Químicos da Água/análise
16.
Sci Total Environ ; 856(Pt 1): 158937, 2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36167130

RESUMO

Atmospheric mercury (Hg) is largely assimilated by vegetation and subsequently transferred to the soil by litterfall, which highlights the role of forests as one of the largest global Hg sinks within terrestrial ecosystems. We assessed the pool of Hg in the aboveground biomass (leaves, wood, bark, branches and twigs), the Hg deposition flux through litterfall over two years (by sorting fallen biomass in leaves, twigs, reproductive structures and miscellaneous) and its accumulation in the soil profile in a deciduous forest dominated by Betula alba from SW Europe. The total Hg pool in the aboveground birch biomass was in the range 532-683 mg ha-1, showing the following distribution by plant tissues: well-developed leaves (171 mg ha-1) > twigs (160 mg ha-1) > bark (159 mg ha-1) > bole wood (145 mg ha-1) > fine branches (25 mg ha-1) > thick branches (24 mg ha-1) > newly sprouted leaves (20 mg ha-1). The total Hg deposition fluxes through litterfall were 15.4 and 11.7 µg m-2 yr-1 for the two years studied, with the greatest contribution coming from birch leaves (73 %). In the soil profile, the pool of Hg in the mineral soil (37.0 mg m-2) was an order of magnitude higher than in the organic horizons (1.0 mg m-2), mostly conditioned by parameters such as soil bulk density and thickness, total C and N contents and the presence of certain Al compounds.


Assuntos
Mercúrio , Árvores , Árvores/química , Mercúrio/análise , Solo , Betula , Ecossistema , Biomassa , Monitoramento Ambiental , Florestas , Betulaceae
17.
Sci Total Environ ; 904: 167532, 2023 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-37797758

RESUMO

Soils constitute the major reservoir of mercury (Hg) in terrestrial ecosystems, whose stability may be threatened by wildfires. This research attempts to look at the effect of prescribed fire on the presence of Hg in a shrubland ecosystem from NE Portugal, delving into its relationship with soil aggregate size and the molecular composition of soil organic matter (SOM). During the prescribed fire, on average 347 mg Hg ha-1 were lost from the burnt aboveground biomass of shrubs and 263 mg Hg ha-1 from the combustion of the soil organic horizon. Overall, Hg concentration and pools in the mineral soil did not show significant changes due to burning, which highlights their role as long-term Hg reservoirs. The higher Hg concentrations found in smaller aggregates (<0.2 mm) compared to coarser ones (0.5-2 mm) are favored by the higher degree of organic matter decomposition (low C/N ratio), rather than by greater total organic C contents. The Hg-enriched finest fraction of soil (<0.2 mm) could be more prone to be mobilized by erosion, whose potential arrival to water bodies increases the environmental concern for the Hg present in fire-affected soils. The SOM quality (molecular composition) and the main organic families, analyzed by Fourier-transform infrared spectroscopy in combination with multivariate statistical analysis, significantly conditioned the retention/emission of Hg in the uppermost soil layers. Thus, before the fire, Hg was strongly linked to lipid and protein fractions, while Hg appeared to be linked to aromatic-like compounds in fire-affected SOM.

19.
MicroPubl Biol ; 20222022.
Artigo em Inglês | MEDLINE | ID: mdl-35088044

RESUMO

Herein, we tested the ability of UNC-33L to rescue dauer formation, lifespan, and locomotion defects of unc-33(mn407) mutants. Results show that the presence of UNC-33L does not rescue the defective dauer phenotype in unc-33(mn407) mutants. However, UNC-33L significantly rescued premature death and uncoordinated locomotion in young unc-33(mn407) adults. The degree of UNC-33L-mediated rescue was less noticeable as the nematodes aged, denoting that both age and the presence of UNC-33L interact in the production of the phenotypes.

20.
Sci Rep ; 12(1): 2417, 2022 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-35165339

RESUMO

Programmed death ligand 1 (PD-L1) plays a key role in glioblastoma multiforme (GBM) immunosuppression, vitality, proliferation, and migration, and is therefore a promising target for treating GBM. CRISPR/Cas9-mediated genomic editing can delete both cell surface and intracellular PD-L1. This systemic deliverable genomic PD-L1 deletion system can be used as an effective anti-GBM therapy by inhibiting tumor growth and migration, and overcoming immunosuppression. To target PD-L1 for CRISPR/Cas9 gene editing, we first identified two single guide RNA (sgRNA) sequences located on PD-L1 exon 3. The first sgRNA recognizes the forward strand of human PD-L1 near the beginning of exon 3 that allows editing by Cas9 at approximately base pair 82 (g82). The second sgRNA recognizes the forward strand of exon 3 that directs cutting at base pair 165 (g165). A homology-directed repair template (HDR) combined with the dual-sgRNAs was used to improve PD-L1 knockout specificity and efficiency. sgRNAs g82 and g165 were cloned into the multiplex CRISPR/Cas9 assembly system and co-transfected with the HDR template in human U87 GBM cells (g82/165 + HDR). T7E1 analysis suggests that the dual-sgRNA CRISPR/Cas9 strategy with a repair template was capable of editing the genomic level of PD-L1. This was further confirmed by examining PD-L1 protein levels by western blot and immunofluorescence assays. Western blot analysis showed that the dual-sgRNAs with the repair template caused a 64% reduction of PD-L1 protein levels in U87 cells, while immunostaining showed a significant reduction of intracellular PD-L1. PD-L1 deletion inhibited proliferation, growth, invasion and migration of U87 cells, indicating intracellular PD-L1 is necessary for tumor progression. Importantly, U87 cells treated with g82/165 + HDR polarized tumor-associated macrophages (TAM) toward an M1 phenotype, as indicated by an increase in TNF-α and a decrease in IL-4 secretions. This was further confirmed with flow cytometry that showed an increase in the M1 markers Ly6C + and CD80 +, and a decrease in the M2 marker CD206 + both in vitro and in vivo. Utilizing dual-sgRNAs and an HDR template with the CRISPR/Cas9 gene-editing system is a promising avenue for the treatment of GBM.


Assuntos
Antígeno B7-H1/genética , Polaridade Celular , Glioblastoma/genética , Glioblastoma/fisiopatologia , Macrófagos Associados a Tumor/citologia , Antígeno B7-H1/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proliferação de Células , Éxons , Edição de Genes , Técnicas de Silenciamento de Genes , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Interleucina-4/genética , Interleucina-4/metabolismo , Invasividade Neoplásica , RNA Guia de Cinetoplastídeos , Macrófagos Associados a Tumor/metabolismo
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