Selection of internalizing RNA aptamers into human breast cancer cells derived from primary sites.

Breast cancer is the most common cancer in women. Although chemotherapy is still broadly used in its treatment, adverse effects remain a challenge. In this scenario, aptamers emerge as a promising alternative for theranostic applications. Studies using breast cancer cell lines provide useful information in laboratory and preclinical investigations, most of which use cell lines established from metastatic sites. However, these cell lines correspond to cell populations of the late stage of tumor progression. On the other hand, studies using breast cancer cells established from primary sites make it possible to search for new theranostic approaches in the early stages of the disease. Therefore, this work aimed to select RNA aptamers internalized by MGSO-3 cells, a human breast cancer cell line, derived from a primary site previously established in our laboratory. Using the Cell-Internalization SELEX method, we have selected two candidate aptamers (ApBC1 and ApBC2). We evaluated their internalization efficiencies, specificities, cellular localization by Reverse Transcription-qPCR (RT-qPCR) and confocal microscopy assays. The results suggest that both aptamers were efficiently internalized by human breast cancer cells, MACL-1, MDA-MB-231, and especially by MGSO-3 cells. Furthermore, both aptamers could effectively distinguish human breast cancer cells derived from normal human mammary cell (MCF 10A) and prostate cancer cell (PC3) lines. Therefore, ApBC1 and ApBC2 could be promising candidate molecules for theranostic applications, even in the early stages of tumor progression.

Spider venom neurotoxin based bioinsecticides: A novel bioactive for the control of the Asian citrus psyllid Diaphorina citri (Hemiptera).

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is a key vector of the phloem-limited bacteria Candidatus Liberibacter asiaticus (CLas) associated with huanglongbing (HLB), the most serious and currently incurable disease of citrus worldwide. Here we report the first investigation into the potential use of a spider venom-derived recombinant neurotoxin, ω/κ-HxTx-Hv1h (hereafter HxTx-Hv1h) when delivered alone or when fused to snowdrop lectin (Galanthus nivalis agglutinin; GNA) to control D. citri. Proteins, including GNA alone, were purified from fermented transformed yeast Pichia pastoris cultures. Recombinant HxTx-Hv1h, HxTx-Hv1h/GNA and GNA were all orally toxic to D. citri, with Day 5 median lethal concentrations (LC50) derived from dose-response artificial diet assays of 27, 20 and 52 µM, respectively. Western analysis of whole insect protein extracts confirmed that psyllid mortality was attributable to protein ingestion and that the fusion protein was stable to cleavage by D. citri proteases. When applied topically (either via droplet or spray) HxTx-Hv1h/GNA was the most effective of the proteins causing >70 % mortality 5 days post treatment, some 2 to 3-fold higher levels of mortality as compared to the toxin alone. By contrast, no significant mortality or phenotypic effects were observed for bumble bees (Bombus terrestris L.) fed on the recombinant proteins in acute toxicity assays. This suggests that HxTx-Hv1h/GNA has potential as a novel bioinsecticide for the management of D. citri offering both enhanced target specificity as compared to chemical pesticides and compatibility with integrated pest management (IPM) strategies.

The Phenotypic Spectrum of Spinocerebellar Ataxia Type 19 in a Series of Latin American Patients.

Spinocerebellar ataxia 19 (SCA19) represents a rare autosomal dominant genetic disorder resulting in progressive ataxia and cerebellar atrophy. SCA19 is caused by variants in the KCND3 gene, which encodes a voltage-gated potassium channel subunit essential for cerebellar Purkinje cell function. We describe six cases from Chile and Mexico, representing the largest report on SCA19 in Latin America. These cases encompass a range of clinical presentations, highlighting the phenotypic variability within SCA19 from an early-onset, severe disease to a late-onset, slowly progressive condition with normal lifespan. While some patients present with pure ataxia, others also show cognitive impairment, dystonia, and other neurological symptoms. The correlations between specific KCND3 variants and phenotypic outcomes are complex and warrant further investigation. As the genomic landscape of spinocerebellar ataxias evolves, comprehensive genetic testing is becoming pivotal in improving diagnostic accuracy. This study contributes to a better understanding of the clinical spectrum of SCA19, laying the groundwork for further genotype-phenotype correlations and functional studies to elucidate the underlying pathophysiology.

The greening-causing agent alters the behavioral and electrophysiological responses of the Asian citrus psyllid to a putative sex pheromone.

The Asian Citrus Psyllid (ACP), Diaphorina citri, is a vector of the pathological bacterium Candidatus Liberibacter asiaticus (CLas), which causes the most devastating disease to the citrus industry worldwide, known as greening or huanglongbing (HLB). Earlier field tests with an acetic acid-based lure in greening-free, 'Valencia' citrus orange groves in California showed promising results. The same type of lures tested in São Paulo, Brazil, showed unsettling results. During the unsuccessful trials, we noticed a relatively large proportion of females in the field, ultimately leading us to test field-collected males and females for Wolbachia and CLas. The results showed high rates of Wolbachia and CLas infection in field populations. We then compared the olfactory responses of laboratory-raised, CLas-free, and CLas-infected males to acetic acid. As previously reported, CLas-uninfected males responded to acetic acid at 1 µg. Surprisingly, CLas-infected males required 50 × higher doses of the putative sex pheromone, thus explaining the failure to capture CLas-infected males in the field. CLas infection was also manifested in electrophysiological responses. Electroantennogram responses from CLas-infected ACP males were significantly higher than those obtained with uninfected males. To the best of our knowledge, this is the first report of a pathogen infection affecting a vector's response to a sex attractant.

Performance of collagen-based matrices from Nile tilapia skin: A pilot proteomic study in a murine model of wound healing.

Full-thickness cutaneous trauma, due to the lack of dermis, leads to difficulty in epithelialization by keratinocytes, developing a fibrotic scar, with less elasticity than the original skin, which may have disorders in predisposed individuals, resulting in hypertrophic scar and keloids. Biomedical materials have excellent characteristics, such as good biocompatibility and low immunogenicity, which can temporarily replace traditional materials used as primary dressings. In this work, we developed two dermal matrices based on Nile tilapia collagen, with (M_GAG) and without (M) glycosaminoglycans, using a sugarcane polymer membrane as a matrix support. To assess the molecular mechanisms driving wound healing, we performed qualitative proteomic analysis on the wound bed in an in vivo study involving immunocompetent murine models at 14 and 21 days post-full-thickness skin injury. Gene Ontology and Pathway analysis revealed that both skins were markedly represented by modulation of the immune system, emphasizing controlling the acute inflammation response at 14 and 21 days post-injury. Furthermore, both groups showed significant enrichment of pathways related to RNA and protein metabolism, suggesting an increase in protein synthesis required for tissue repair and proper wound closure. Other pathways, such as keratinization and vitamin D3 metabolism, were also enriched in the groups treated with M matrix. Finally, both matrices improved wound healing in a full post-thick skin lesion. However, our preliminary molecular data reveals that the collagen-mediated healing matrix lacking glycosaminoglycan (M) exhibited a phenotype more favorable to tissue repair, making it more suitable for use before skin grafts.

Targeting ABCA12-controlled ceramide homeostasis inhibits breast cancer stem cell function and chemoresistance.

Cancer stem cells (CSCs) drive tumor growth, metastasis, and chemoresistance. While emerging evidence suggests that CSCs have a unique dependency on lipid metabolism, the functions and regulation of distinct lipid species in CSCs remain poorly understood. Here, we developed a stem cell factor SOX9-based reporter for isolating CSCs in primary tumors and metastases of spontaneous mammary tumor models. Transcriptomic analyses uncover that SOX9high CSCs up-regulate the ABCA12 lipid transporter. ABCA12 down-regulation impairs cancer stemness and chemoresistance. Lipidomic analyses reveal that ABCA12 maintains cancer stemness and chemoresistance by reducing intracellular ceramide abundance, identifying a CSC-associated function of ABCA subfamily transporter. Ceramide suppresses cancer stemness by inhibiting the YAP-SOX9 signaling pathway in CSCs. Increasing ceramide levels in tumors enhances their sensitivity to chemotherapy and prevents the enrichment of SOX9high CSCs. In addition, SOX9high and ABCA12high cancer cells contribute to chemoresistance in human patient-derived xenografts. These findings identify a CSC-suppressing lipid metabolism pathway that can be exploited to inhibit CSCs and overcome chemoresistance.

Widespread synchronous decline of Mediterranean-type forest driven by accelerated aridity.

Large-scale, abrupt ecosystem change in direct response to climate extremes is a critical but poorly documented phenomenon1. Yet, recent increases in climate-induced tree mortality raise concern that some forest ecosystems are on the brink of collapse across wide environmental gradients2,3. Here we assessed climatic and productivity trends across the world's five Mediterranean forest ecosystems from 2000 to 2021 and detected a large-scale, abrupt forest browning and productivity decline in Chile (>90% of the forest in <100 days), responding to a sustained, acute drought. The extreme dry and warm conditions in Chile, unprecedented in the recent history of all Mediterranean-type ecosystems, are akin to those projected to arise in the second half of the century4. Long-term recovery of this forest is uncertain given an ongoing decline in regional water balance. This dramatic plummet of forest productivity may be a spyglass to the future for other Mediterranean ecosystems.

Density and Dispersal Radius of Tamarixia radiata for Control of Diaphorina citri in Citrus Groves.

Tamarixia radiata (Waterston) is the main parasitoid wasp released in abandoned, organic, and non-commercial citrus groves and in orange jasmine areas  for biological control of Diaphorina citri Kuwayama (a vector of bacteria associated with huanglongbing), as a control tactic in a strategy termed External Management. The effectiveness of parasitism is influenced by the release density and radius of action of this parasitoid. This study determined the optimal release density and radius of action of T. radiata in a commercial citrus grove. Parasitoids were released at five densities (200, 400, 800, 1,600, and 3,200 wasps/ha) at the center of circular areas with four different radii (7.5, 15, 22.5, and 30 m). The parasitism rate was assessed using 'Valencia' sweet orange seedlings, each bearing 35 D. citri fourth-instar nymphs, attached to 26-year-old 'Valencia' sweet orange trees along the perimeter of each radius. For the 7.5-m radius, the highest parasitism rate (74.1%) was observed at the density of 3,200 wasps/ha. However, for the 15-m radius, the density of 3,200 wasps/ha resulted in lower parasitism (30.7%) of D. citri nymphs. No parasitism was observed for densities lower than 800 wasps/ha and release radii above 15 m. The highest parasitoid dispersal radius and area were 18.04 m and 114.02 m2, respectively, when 3,200 wasps were released. Therefore, 3,200 wasps/ha arranged at 56 equidistant release points (approximately 57 wasps/release point) is the recommended protocol for biological control of D. citri in citrus groves.

An Exploratory Survey on the Care for Ataxic Patients in the American Continents and the Caribbean.

Little is known about access of rare disease carriers to health care. To increase this knowledge, the Pan American Hereditary Ataxia Network (PAHAN) conducted an exploratory survey about care for hereditary ataxias in American continents and the Caribbean. A questionnaire was sent to health professionals about the hereditary ataxias identified; access to care; and local teaching and research. The number of ataxics under current care per 100,000 inhabitants was subtracted from the expected overall prevalence of 6/100,000, to estimate the prevalence of uncovered ataxic patients. Local Human Development Indexes (HDI) were used to measure socio-economic factors. Twenty-six sites participated. Twelve sites had very high, 13 had high, and one site had medium HDI. Participants reported on 2239 and 602 patients with spinocerebellar ataxias and recessive forms under current care. The number of patients under current care per inhabitants varied between 0.14 and 12/100,000. The estimated prevalence of uncovered ataxic patients was inversely proportional to HDIs (rho = 0.665, p = 0.003). Access to diagnosis, pre-symptomatic tests, and rehabilitation were associated with HDIs. More and better molecular diagnostic tools, protocols and guidelines, and professional training for ataxia care were the top priorities common to all respondents. Evidence of inequalities was confirmed. Lower HDIs were associated with high potential numbers of uncovered ataxic subjects, and with lack of molecular diagnosis, pre-symptomatic testing, and rehabilitation. More and better diagnostic tools, guidelines, and professional training were priorities to all sites. PAHAN consortium might help with the last two tasks.

Resistance of True Citrus species to Diaphorina citri.

BACKGROUND: Host genetic resistance is a promising strategy for the management of Diaphorina citri Kuwayama (Hemiptera: Psyllidae), and consequently Huanglongbing (HLB). To date, no study has investigated the resistance to D. citri in the clonal and vegetatively propagated plants of the Microcitrus, Eremocitrus, and Atalantia genera. This study assesses Near and True Citrus genotype antixenosis and antibiosis against D. citri, with trichome density and volatile emission as possible mechanisms of resistance. RESULTS: All genotypes were oviposited by D. citri, however, 8 of 14 genotypes were less oviposited than Citrus × sinensis 'Valencia' (susceptible control). Diaphorina citri nymphs had lower nymphal viability in E. glauca (31%) and M. warburgiana (58%) than that in Citrus × sinensis (77%). The behavioral assay showed that 30% of D. citri nymphs in the last instars evaded E. glauca shoots, whereas no nymphs evaded Citrus × sinensis shoots. A higher trichome density was observed in E. glauca shoots compared to the other genotypes. Chemical analysis revealed differences in the volatile profiles of E. glauca and Citrus × sinensis. CONCLUSION: Eremocitrus glauca and M. warburgiana genotypes were more resistant to D. citri than Citrus × sinensis. Higher trichome density in the shoots may negatively influence the development of D. citri nymphs. Eremocitrus glauca volatiles may also be involved in their resistance to D. citri. © 2022 Society of Chemical Industry.

Gastroschisis prognostic score successfully identifies Brazilian newborns with high-risk gastroschisis.

BACKGROUND: The Gastroschisis Prognostic Score (GPS) stratifies patients as high or low risk based on the visual assessment of intestinal matting, atresia, perforation, or necrosis. Despite being a simple score, its applicability to low and middle-income countries (LMICs) remains unknown. We tested the hypothesis that GPS can predict outcomes in LMICs, by assessing the prognostic value of the GPS in a middle-income country. METHODS: This prospective study followed all newborns with gastroschisis in a Brazilian neonatal unit based in a public hospital from 2015-2019. Infants were stratified into low and high-risk cohorts based on the GPS. In addition to basic demographics, data collected included duration of parenteral nutrition (TPN), mechanical ventilation (MV), length of stay (LOS), suspicion of infection that led to the use of antibiotics, and mortality. Univariate and multivariate analyses were conducted to identify which outcomes the GPS independently predicted. RESULTS: Sixty-one newborns with gastroschisis were treated during the study period. The mean birth weight, gestational age, and 5' Apgar score were 2258 g, 36 weeks, and 9. Twenty-four infants (39.3%) were identified as low-risk (GPS < 2) and 37 (60.7%) as high-risk (GPS > 2). The high-risk group presented with prolonged TPN use (p<0.001), MV (p<0.001), and LOS (p:0.002). GPS did not predict antibiotic therapy or mortality. CONCLUSION: In the first study in a middle-income country, the GPS predicted several important clinical outcomes. The GPS is a reliable tool for parental counseling and resource allocation in diverse settings. LEVEL OF EVIDENCE: II.c (cohort prospective).

Impact of diseases and pests on premature fruit drop in sweet orange orchards in São Paulo state citrus belt, Brazil.

BACKGROUND: Despite technical improvements in the citrus chain and leadership in orange production achieved in the past decades, premature fruit drop remains a major component of crop loss in São Paulo state citrus belt, the largest sweet orange production area in the world. The present study aimed to determine, during five consecutive seasons, the impact of the diseases and pests on premature fruit drop in the orange belt. RESULTS: Fruit drop due to the main diseases and pests averaged approximately 11.0%, which corresponded to approximately 63% of the annual fruit drop. The average fruit drop rate due to fruit borer and fruit flies combined was 4.0%, Huanglongbing (HLB) 3.3%, black spot 2.6%, leprosis 1.0% and citrus canker 0.3%. The average amount of fruit drop (million 40.8 kg boxes) and value of crop losses (million US$ dollars), in five seasons, were 12.7 and 66.2 for fruit borer/fruit flies, 11.0 and 57.9 for HLB, 8.1 and 42.2 for black spot, 3.1 and 15.6 for leprosis, and 0.9 and 4.9 for citrus canker, respectively. CONCLUSION: Fruit borer and fruit flies (combined), HLB, black spot, leprosis and citrus canker are, in this order, the main diseases and pests in the orange belt of São Paulo state. All of these causes significantly increased the overall fruit drop rate in the evaluated seasons. The results will contribute to the development of the Brazilian citrus industry, while showing to other citrus-growing regions the potential that diseases and pests have to jeopardize production. © 2022 Society of Chemical Industry.

Cultural Management of Huanglongbing: Current Status and Ongoing Research.

Huanglongbing (HLB), formerly known as greening, is a bacterial disease restricted to some Asian and African regions until two decades ago. Nowadays, associated bacteria and their vectors have spread to almost all citrus-producing regions, and it is currently considered the most devastating citrus disease. HLB management can be approached in terms of prevention, limiting or avoiding pathogen and associated vectors to reach an area, or in terms of control, trying to reduce the impact of the disease by adopting different cultural strategies depending on infestation/infection levels. In both cases, control of psyllid populations is currently the best way to stop HLB spread. Best cultural actions (CHMAs, TPS system) to attain this goal and, thus, able to limit HLB spread, and ongoing research in this regard is summarized in this review.

Evaluation of Masseter Muscle Dimensions Through Ultrasonography

ABSTRACT: The objective this study was to determine mean values of masseter muscle thickness in male and female, at rest and contraction, in healthy individuals. At first, 102 questionnaires were distributed between students. Thirty patients were selected, 15 male and 15 female, according to the inclusion criteria, excluding all individuals with symptoms of temporo-mandibular dysfunction or syndromes with craniofacial disorders. Masseter muscle thickness was determined bilaterally, at rest and contraction. As a reference point for the measurement of thickness (axial plane), the posterior prolon gation of the labial commissure was used. Normality values of the masseter muscle in females were 11.75 ± 1,14 mm in contraction and 10.19 ± 1.04 mm in relaxation; and in the male were 13.95 ± 1.62 mm in contraction and 11.64 mm ± 1.68 mm in relaxation. The results obtained are in agreement with those described in the literature. Demonstrating the accuracy and reproductibility of the ultrasound if a protocol is applied, mainly aimed at controlling the pressure applied by the transducer.

RESUMEN: El objetivo de este estudio fue determinar los valores medios del grosor del músculo masetero, en reposo y contracción, en hombres y mujeres sanos. Inicialmente se distribuyeron 102 cuestionarios entre los estudiantes. Se seleccionaron 30 pacientes, 15 hombres y 15 mujeres, según los criterios de inclusión, excluyendo a todos los individuos con síntomas de disfunción temporo-mandibular o síndromes con alteraciones craneofaciales. El grosor del músculo masetero se determinó de forma bilateral, en reposo y contracción. Como punto de referencia para la medición del espesor (plano axial), la prolongación posterior de la comisura labial. Los valores de normalidad del músculo masetero en las mujeres fue de 11,75 ± 1,14 mm en contracción y 10,19 ± 1,04 mm en relajación; y en los varones 13,95 ± 1,62 mm en contracción y 11,64 mm ± 1,68 mm en relajación. Los resultados obtenidos coinciden con los descritos en la literatura, señalando la precisión y reproducibilidad del ultrasonido si se aplica un protocolo, principalmente dirigido a controlar la presión aplicada por el transductor.

Roles of mesenchymal stromal cells in the head and neck cancer microenvironment.

Head and neck cancer (HNC), a common malignancy worldwide, is associated with high morbidity and mortality rates. Squamous cell carcinoma is the most common HNC type, followed by salivary gland carcinomas, head and neck sarcomas, and lymphomas. The microenvironment of HNCs comprises various cells that regulate tumor development. Recent studies have reported that the tumor microenvironment, which modulates cancer progression, regulates cancer treatment response. However, the presence of different types of stromal cells in cancers is a major challenge to elucidate the role of individual cells in tumor progression. The role of mesenchymal stromal cells (MSCs), which are a component of the tumor microenvironment, in HNC is unclear. The major impediment for characterizing the role of MSCs in cancer progression is the lack of MSC-specific markers and their phenotypic similarity with stromal cells. This review aimed to summarize the latest findings on the role of MSCs in the progression of HNC to improve our understanding of HNC pathophysiology.

The basis of nuclear phospholipase C in cell proliferation.

Ca2+ is a highly versatile intracellular signal that regulates many biological processes such as cell death and proliferation. Broad Ca2+-signaling machinery is used to assemble signaling systems with a precise spatial and temporal resolution to achieve this versatility. Ca2+-signaling components can be organized in different regions of the cell and local increases in Ca2+ within the nucleus can regulate different cellular functions from the increases in cytosolic Ca2+. However, the mechanisms and pathways that promote localized increases in Ca2+ levels in the nucleus are still under investigation. This review presents evidence that the nucleus has its own Ca2+ stores and signaling machinery, which modulate processes such as cell proliferation and tumor growth. We focus on what is known about the functions of nuclear Phospholipase C (PLC) in the generation of nuclear Ca2+ transients that are involved in cell proliferation.

Nikolai Gogol's account of sleep paralysis in the tale "The Portrait".

Several classical writers had an impressive power of observation and often depicted medical conditions in their works long before medical literature did. Sleep paralysis is a common and frightening experience, in particular when occurring for the first time. Therefore, it is not surprising that it has been frequently described in the classical literature, eg by Dostoevsky, Kafka, Dickens, and Maupassant. In Nikolai Gogol's tale "The portrait" (1833) we could recognize an excellent description of a sleep paralysis, in which several components of this condition were depicted including motor paralysis, visual and auditory hallucinations, and autonomic manifestations. To the best of our knowledge, this account is the earliest description of a sleep paralysis in non-medical literature.

Adipose-derived stem/stromal cell secretome modulates breast cancer cell proliferation and differentiation state towards aggressiveness.

It is becoming increasingly evident that mesenchymal stem/stromal cells are recruited by cancer cells from nearby endogenous host stroma and promote events such as tumor proliferation, angiogenesis, invasion, and metastasis, as well as mediate therapeutic resistance. Consequently, understanding the regulatory mechanisms of ASCs that influence the tumor microenvironment may provide an avenue for further treatment. To understand the role of the ASC secretome in breast cancer cell proliferation, death, and phenotype alteration, adipose-derived stem cell-conditioned medium (mASC) was used to cultivate MCF-7 and MDA-MB-231 cells. These breast cancer cells in mASC showed a shorter doubling time, higher frequency of EdU positivity, and higher levels of phosphorylated histone 3. In addition, increased expression of cyclin B1 was observed, suggesting that proliferation was induced. The mASC was also able to increase apoptosis in MCF-7 cells, which was confirmed by caspase-7 activation. The number of tumor-initiating cells (CD44+ CD24-/low) and migration capacity were increased in cells cultivated in mASC. These data collectively suggest that ASC-conditioned medium can induce selective pressure by increasing cell proliferation, giving rise to a more aggressive phenotype in MCF-7 and MDA-MB-231 cells. Our study provides a foundation for further elucidation of the precise mechanism underlying ASCs in breast cancer cells and the modulation of ASCs in potential therapeutic uses.

Selection of DNA Aptamers for Differentiation of Human Adipose-Derived Mesenchymal Stem Cells from Fibroblasts.

In recent years, stem cell therapy has shown promise in regenerative medicine. The lack of standardized protocols for cell isolation and differentiation generates conflicting results in this field. Mesenchymal stem cells derived from adipose tissue (ASC) and fibroblasts (FIB) share very similar cell membrane markers. In this context, the distinction of mesenchymal stem cells from fibroblasts has been crucial for safe clinical application of these cells. In the present study, we developed aptamers capable of specifically recognize ASC using the Cell-SELEX technique. We tested the affinity of ASC aptamers compared to dermal FIB. Quantitative PCR was advantageous for the in vitro validation of four candidate aptamers. The binding capabilities of Apta 2 and Apta 42 could not distinguish both cell types. At the same time, Apta 21 and Apta 99 showed a better binding capacity to ASC with dissociation constants (Kd) of 50.46 ± 2.28 nM and 72.71 ± 10.3 nM, respectively. However, Apta 21 showed a Kd of 86.78 ± 9.14 nM when incubated with FIB. Therefore, only Apta 99 showed specificity to detect ASC by total internal reflection microscopy (TIRF). This aptamer is a promising tool for the in vitro identification of ASC. These results will help understand the differences between these two cell types for more specific and precise cell therapies.