Advances in Tissue Culture and Transformation Studies in Non-model Species: Bixa orellana L. (Bixaceae).

Over the years, our team has dedicated significant efforts to studying a unique natural dye-producing species, annatto (Bixa orellana L.). We have amassed knowledge and established foundations that support the applications of gene expression analysis in comprehending in vitro morphogenic regeneration processes, phase transition aspects, and bixin biosynthesis. Additionally, we have conducted gene editing associated with these processes. The advancements in this field are expected to enhance breeding practices and contribute to the overall improvement of this significant woody species. Here, we present a step-by-step protocol based on somatic embryogenesis and an optimized transformation protocol utilizing Agrobacterium tumefaciens.

Dynamics of annatto pigment synthesis and accumulation in seeds of Bixa orellana L. revealed by integrated chemical, anatomical, and RNA-Seq analyses.

Bixin is a commercially valuable apocarotenoid pigment found in the seed aril of Bixa orellana. The dynamics and regulation of its biosynthesis and accumulation during seed development remain largely unknown. Here, we combined chemical, anatomical, and transcriptomic data to provide stage-specific resolution of the cellular and molecular events occurring during B. orellana seed development. Seeds at five developmental stages (S1-S5) were used for analysis of bixin content and seed anatomy, and three of them (S1, S3, and S4) were selected for Illumina HiSeq sequencing. Bixin accumulated in large quantities in seeds compared with other tissues analyzed, particularly during the S2 stage, peaking at the S4 stage, and then decreasing slightly in the S5 stage. Anatomical analysis revealed that bixin accumulated in the large central vacuole of specialized cells, which were scattered throughout the developing mesotesta at the S2 stage, but enlarged progressively at later stages, until they occupied most of the parenchyma in the aril. A total of 13 million reads were generated and assembled into 73,381 protein-encoding contigs, from which 312 were identified as containing 1-deoxy-D-xylulose-5-phosphate/2-C-methyl-D-erythritol-4-phosphate (DOXP/MEP), carotenoid, and bixin pathways genes. Differential transcriptome expression analysis of these genes revealed that 50 of them were sequentially and differentially expressed through the seed developmental stages analyzed, including seven carotenoid cleavage dioxygenases, eight aldehyde dehydrogenases, and 22 methyltransferases. Taken together, these results show that bixin synthesis and accumulation in seeds of B. orellana are a developmentally regulated process involving the coordinated expression of DOXP/MEP, carotenoid, and bixin biosynthesis genes.

Intensive calcium monitoring improves outcomes on hungry bone syndrome in hyperparathyroidism.

Objectives. Hungry bone syndrome (HBS) is a severe and underdiagnosed complication of parathyroidectomy in the treatment of primary hyperparathyroidism (PHP) and secondary hyper-parathyroidism to chronic kidney disease (SHP-CKD).Methods. A longitudinal study was conducted to compare the postoperative outcomes of patients who developed HBS in two different time frames: before and after implementing a protocol with an intensive electrolytic monitoring and an algorithm regarding electrolytic supplementation.Results. Overall, 77 parathyroidectomies were included. In PHP, a protocol implementation led to an increased admission of patients in the Intermediate Care Unit for intensive electrolytic monitoring (p<0.001) and an increased rate of oral calcium replacement during hospital stay (p=0.013) compared to pre-protocol era. In SHP-CKD, duration of intravenous calcium replacement was reduced (p=0.010). The prevalence of HBS (9.8% in PHP and 58.3% in SHP-CKD) was similar between the two periods, although its diagnosis had an increased trend in PHP since the protocol implementation. None of the diagnosis of HBS was established due to hypocalcemic symptoms in the post-protocol era (contrary to pre-protocol period, p=0.021). Both hypocalcemia length and duration of surgical ward hospitalization were reduced (p=0.047 and p=0.042, respectively).Conclusions. An improved assessment of hyperparathyroidism and a decrease in HBS severity were noted in the post-protocol era. We strongly recommend the implementation of a standardized protocol with an intensive phosphocalcium monitoring in the high-risk patients who undergo parathyroidectomy due to hyperparathyroidism as it improves the health care and management of HBS.

High-refined carbohydrate diet consumption induces neuroinflammation and anxiety-like behavior in mice.

Consumption of poor nutrients diets is associated with fat tissue expansion and with a central and peripheral low-grade inflammation. In this sense, the microglial cells in the central nervous system are activated and release pro-inflammatory cytokines that up-regulate the inducible nitric oxide synthase (iNOS), promoting Nitric Oxide (NO) production. The excess of NO has been proposed to facilitate anxious states in humans and rodents. We evaluated whether consumption of a high-refined carbohydrate-containing diet (HC) in mice induced anxiety-like behavior in the Novelty Suppressed Feeding Test (NFST) trough facilitation of NO, in the prefrontal cortex (PFC) and hippocampus (HIP). We also verified if HC diet induces activation of microglial cells, alterations in cytokine and leptin levels in such regions. Male BALB/c mice received a standard diet or a HC diet for 3 days or 12 weeks. The chronic consumption of HC diet, but not acute, induced an anxiogenic-like effect in the NSF test and an increase in the nitrite levels in the PFC and HIP. The preferential iNOS inhibitor, aminoguanidine (50 mg/kg, i.p.), attenuated such effects. Moreover, microglial cells in the HIP and PFC were activated after chronic consumption of HC diet. Finally, the expression of iNOS in the PFC and TNF, IL6 and leptin levels in HIP were higher in chronically HC fed mice. Taken together, our data reinforce the notion that diets containing high-refined carbohydrate facilitate anxiety-like behavior, mainly after a long period of consumption. The mechanisms involve, at least in part, the augmentation of neuroinflammatory processes in brain areas responsible for anxiety control.

Selection and validation of reference genes for quantitative gene expression analyses in various tissues and seeds at different developmental stages in Bixa orellana L.

Bixa orellana L., popularly known as annatto, produces several secondary metabolites of pharmaceutical and industrial interest, including bixin, whose molecular basis of biosynthesis remain to be determined. Gene expression analysis by quantitative real-time PCR (qPCR) is an important tool to advance such knowledge. However, correct interpretation of qPCR data requires the use of suitable reference genes in order to reduce experimental variations. In the present study, we have selected four different candidates for reference genes in B. orellana, coding for 40S ribosomal protein S9 (RPS9), histone H4 (H4), 60S ribosomal protein L38 (RPL38) and 18S ribosomal RNA (18SrRNA). Their expression stabilities in different tissues (e.g. flower buds, flowers, leaves and seeds at different developmental stages) were analyzed using five statistical tools (NormFinder, geNorm, BestKeeper, ΔCt method and RefFinder). The results indicated that RPL38 is the most stable gene in different tissues and stages of seed development and 18SrRNA is the most unstable among the analyzed genes. In order to validate the candidate reference genes, we have analyzed the relative expression of a target gene coding for carotenoid cleavage dioxygenase 1 (CCD1) using the stable RPL38 and the least stable gene, 18SrRNA, for normalization of the qPCR data. The results demonstrated significant differences in the interpretation of the CCD1 gene expression data, depending on the reference gene used, reinforcing the importance of the correct selection of reference genes for normalization.

Autonomously hyperfunctioning cystic nodule harbouring thyroid carcinoma - Case report and literature review.

INTRODUCTION: Hyperthyroidism is rarely associated with malignancy, but it cannot rule out thyroid cancer. Although there is published data describing this coexistence, thyroid carcinomas inside autonomously functioning nodules are uncommon. PRESENTATION OF CASE: A 49-year-old woman presented with a cervical mass, unexplained weight loss and anxiousness, sweating and insomnia. On physical examination, she had a palpable left thyroid nodule. Thyroid function tests showed suppressed TSH (<0,1 uUI/mL), thyroxine 1,44 ng/dL (normal range 0,70-1,48) and triiodothyronine 4,33 pg/mL (normal range 1,71-3,71). Ultrasound imaging revealed a left lobe, 4 cm partial cystic nodule. 99mTC thyroid scintigraphy showed a hyperfunctioning nodule with suppression of the remainder parenchyma. Fine-needle aspiration cytology was nondiagnostic (cystic fluid). The patient was started on thiamazole 5 mg daily with subsequent normalization of thyroid function, but she developed cervical foreign body sensation and a left hemithyroidectomy was performed. Histology showed a 4 cm cystic nodule with a follicular variant papillary carcinoma and the patient underwent completion thyroidectomy, followed by radio-iodine ablation. DISCUSSION: Published literature showed an increased prevalence of autonomously functioning nodules, harbouring thyroid carcinomas in adults. Papillary carcinoma is the most frequently described but the follicular variant is rare. CONCLUSION: Although rare, thyroid cancer is not definitively excluded in hyperthyroid patients and it should always be considered as differential diagnosis.

Primary thyroid lymphoma: A case report and review of the literature.

INTRODUCTION: A rapidly enlarging mass of the anterior compartment of the neck with compressive symptoms may represent, among other diagnosis, a neoplasm of the thyroid gland. PRESENTATION OF CASE: We describe the case of a 59-year-old woman referred to the endocrine surgical unit because of compressive cervical symptoms for 3 months. The cervical ultrasound revealed a sub-sternal goiter with heterogeneous echo structure and the fine-needle aspirating cytology was inconclusive. Given the large impact of symptoms on life quality, she was submitted to a total thyroidectomy. Histological examination of the surgical specimen revealed the presence of a Diffuse Large B Cell Lymphoma of the thyroid. DISCUSSION: Primary thyroid lymphomas are rare and there are few randomized studies for diagnostic and therapeutic guidance. New immunohistochemical and molecular techniques have improved the diagnostic accuracy with corebiopsy limiting the role of surgery. The treatment should first include the control of local disease with radiotherapy and/or surgery combined with chemotherapy to control obscure or disseminated disease. Palliative surgery may be needed to relieve airway compression symptoms. Under these circumstances, surgery should be performed by a specialized surgeon to decrease the associated morbidity. The prognosis of patients depends on the histological classification of the tumor and the stage of the disease. CONCLUSION: Due to the rarity of the disease, each case must be evaluated and treated individually, since there is not a consensual therapeutic approach.

Characterization of the legumains encoded by the genome of Theobroma cacao L.

Legumains are cysteine proteases related to plant development, protein degradation, programmed cell death, and defense against pathogens. In this study, we have identified and characterized three legumains encoded by Theobroma cacao genome through in silico analyses, three-dimensional modeling, genetic expression pattern in different tissues and as a response to the inoculation of Moniliophthora perniciosa fungus. The three proteins were named TcLEG3, TcLEG6, and TcLEG9. Histidine and cysteine residue which are part of the catalytic site were conserved among the proteins, and they remained parallel in the loop region in the 3D modeling. Three-dimensional modeling showed that the propeptide, which is located in the terminal C region of legumains blocks the catalytic cleft. Comparing dendrogram data with the relative expression analysis, indicated that TcLEG3 is related to the seed legumain group, TcLEG6 is related with the group of embryogenesis activities, and protein TcLEG9, with processes regarding the vegetative group. Furthermore, the expression analyses proposes a significant role for the three legumains during the development of Theobroma cacao and in its interaction with M. perniciosa.

Unraveling new genes associated with seed development and metabolism in Bixa orellana L. by expressed sequence tag (EST) analysis.

The tropical tree Bixa orellana L. produces a range of secondary metabolites which biochemical and molecular biosynthesis basis are not well understood. In this work we have characterized a set of ESTs from a non-normalized cDNA library of B. orellana seeds to obtain information about the main developmental and metabolic processes taking place in developing seeds and their associated genes. After sequencing a set of randomly selected clones, most of the sequences were assigned with putative functions based on similarity, GO annotations and protein domains. The most abundant transcripts encoded proteins associated with cell wall (prolyl 4-hydroxylase), fatty acid (acyl carrier protein), and hormone/flavonoid (2OG-Fe oxygenase) synthesis, germination (MADS FLC-like protein) and embryo development (AP2/ERF transcription factor) regulation, photosynthesis (chlorophyll a-b binding protein), cell elongation (MAP65-1a), and stress responses (metallothionein- and thaumatin-like proteins). Enzymes were assigned to 16 different metabolic pathways related to both primary and secondary metabolisms. Characterization of two candidate genes of the bixin biosynthetic pathway, BoCCD and BoOMT, showed that they belong, respectively, to the carotenoid-cleavage dioxygenase 4 (CCD4) and caffeic acid O-methyltransferase (COMT) families, and are up-regulated during seed development. It indicates their involvement in the synthesis of this commercially important carotenoid pigment in seeds of B. orellana. Most of the genes identified here are the first representatives of their gene families in B. orellana.

Isolation and purification of RNA from tissues rich in polyphenols, polysaccharides, and pigments of annatto (Bixa orellana L.).

The tropical plant Bixa orellana L. (annatto) produces an array of natural products, including the pigment bixin used in the food and cosmetics industries. In order to understand the biochemical and molecular basis of the biosynthesis of these natural products, a reliable method for isolating high yields of high-quality RNA is required. Here we described a successful and reproducible method for isolation and purification of high-quantity and high-quality RNA from different tissues of annatto. This protocol overcomes the usual problems associated with large amounts of polyphenols, polysaccharides, pigments, and other secondary metabolites that are not easily removed by conventional extraction procedures. Furthermore, the proposed protocol can be easily carried out in any laboratory and it could also be extended to isolate RNA from other plant species showing similar abundance of compounds that interfere with RNA extractions. The yield and quality of the RNA were monitored by spectrophotometric analysis, separation on agarose gel, Reverse Transcription-Polymerase Chain Reaction (RT-PCR), and construction of a cDNA library.

[Hospitalized children' perceptions on exams]. / Percepção de crianças hospitalizadas sobre realização de exames.

The study had as objective to identify the hospitalized children's perception, in school age, about the accomplishment of exams. The research of descriptive nature was used, prevailing the investigation of qualitative character. The results revealed that the children cooperate with exams despite the fear. The fear was related to the dread of the physical pain, being hurt, or even the unknown. It is considered that the child notes the exam accomplishment as part of his/ her treatment and he/she would like to receive explanations about the procedure.

Percepção de crianças hospitalizadas sobre realização de exames / Percepción de niños hospitalizados sobre realización de examenes / Hospitalized children' perception on exam accomplishment

O estudo teve como objetivo identificar a percepção da criança hospitalizada, em idade escolar, sobre a realização de exames. Foi utilizada a pesquisa de natureza descritiva, predominando a investigação de caráter qualitativo. Os resultados revelaram que as crianças cooperam com os exames apesar do medo. O medo foi relacionado ao temor da dor física, de ser machucado, e ainda, do desconhecido. Considera-se que a criança percebe a realização do exame como parte do seu tratamento e gostaria de receber explicações sobre o procedimento.

The study had as objective to identify the hospitalized children's perception, in school age, about the accomplishment of exams. The research of descriptive nature was used, prevailing the investigation of qualitative character. The results revealed that the children cooperate with exams despite the fear. The fear was related to the dread of the physical pain, being hurt, or even the unknown. It is considered that the child notes the exam accomplishment as part of his/her treatment and he/she would like to receive explanations about the procedure.

El estudio tuvo como objetivo identificar la percepción del niño hospitalizado, en edad de ir a la escuela, sobre la realización de exámenes. Fue usada la investigación de naturaleza descriptiva, prevaleciendo la investigación de carácter cualitativo. Los resultados revelaron que los niños colaboran con los exámenes a pesar del miedo. EI miedo se relacionó con el temor al dolor físico, a ser herido, o aún, a lo desconocido. Se considera que el niño percibe la realización del examen como parte del tratamiento e desearía recibir explicaciones sobre el procedimiento.

Behçet's syndrome, AIDS, cerebral toxoplasmosis: an unusual association

Há raras descriçöes da AIDS associadas a manifestaçöes sugestivas de síndrome de Behçet. Apresentamos o caso de uma jovem senhora que apresentou progressivamente no período de sete anos, critérios clínicos para o diagnóstico de síndrome de Behçet e que ao desenvolver meningoencefalite linfomonocitária recidivante, obserou-se estar infectada com o virus HIV-1. A terapêutica imunodepressora com glicocorticóides em altas doses näo foi satisfatória e a paciente veio a falecer por toxoplasmose cerebral, suspeita por ressonância magnética e confirmada por necrópsia, após sua terceira internaçäo. Níveis baixos de CD4 e da relaçäo CD4/CD8, algumas vezes observadas na síndrome de Behçet ativa, no entanto, mais altos do que comumente se observa na presença desta complicaçäo oportuísta grave, foram elementos que nos levaram a dificuldades diagnósticas, alertando o clínico para a necessidade de interpretar com restriçöes os achados laboratoriais. A falta de conhecimento definitivo do período no qual se infectou pelo HIV-1 possibilita as seguintes consideraçöes: ou estamos diante de síndrome de superposiçäo de distintas enfermidades; ou, entäo, de uma forma inusitada de apresentaçäo evolutiva, mimetizando a síndrome de Behçet, numa enferma infectada por este vírus, que posteriormente, desenvolveu AIDS