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Clinically non-functioning pituitary adenomas (CNFPAs) are the second most frequent sellar tumor among studies on community-dwelling adults. They are characterized by the absence of hormonal hypersecretion syndrome, and patients present with compressive symptoms, such as a headache and visual field defects. Immunohistochemically, most CNFPAs are of gonadotrope differentiation, with only a few of them being truly null cell adenomas. Although these tumors express receptors for one or more hypothalamic releasing hormones, to what extent this has an impact on the biological and clinical behavior of these neoplasms remains to be defined. In this research, we evaluated the basal and hypothalamic secretagogue-stimulated intracellular calcium mobilization in 13 CNFPAs, trying to correlate this response to the phenotypic features of the patients. Our results indicate that the recurrence of a CNFPA correlates positively with cellular responsiveness, as measured by spontaneous intracellular calcium activity and the ability to respond to multiple hypothalamic secretagogues. We conclude that this finding may be a useful tool for predicting the clinicopathologic behavior of CNFPAs, by testing the variation of cellular responsiveness to hypothalamic secretagogues.
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Segunda Neoplasia Primária , Neoplasias Hipofisárias , Adulto , Humanos , Cálcio , Sinalização do Cálcio , Recidiva Local de Neoplasia , Secretagogos , Cálcio da DietaRESUMO
The outbreak of COVID-19, a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, is regarded as the most severe of the documented coronavirus pandemics. The measurement and monitoring of SARS-CoV-2 antibody levels by serological tests are relevant for a better epidemiological and clinical understanding of COVID-19. The aim of this work was to design a method called the SARS-CoV-2 antibody detection method (SARS-CoV-2 AbDM) for fluorescence immunodetection of anti-SARS-CoV-2 IgG and IgM on both plate and microfluidic chip. For this purpose, a system with magnetic beads that immobilize the antigen (S protein and RBD) on its surface was used to determine the presence and quantity of antibodies in a sample in a single reaction. The SARS-CoV-2 AbDM led to several advantages in the performance of the tests, such as reduced cost, possibility of performing isolated or multiple samples, potential of multiplex detection, and capacity to detect whole blood samples without losing resolution. In addition, due to the microfluidic chip in conjunction with the motorized actuated platform, the time, sample quantity, and operator intervention during the process were reduced. All these advantages suggest that the SARS-CoV-2 AbDM has the potential to be developed as a PoC that can be used as a tool for seroprevalence monitoring, allowing a better understanding of the epidemiological and clinical characteristics of COVID-19 and contributing to more effective and ethical decision-making in strategies to fight against the COVID-19 pandemic.
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Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) diagnostic methods have a large potential to effectively detect SARS-CoV-2 with sensitivity and specificity nearing 100%, comparable to quantitative polymerase chain reaction. Yet, there is room for improvement. Commonly, one guide CRISPR RNA (gRNA) is used to detect the virus DNA and activate Cas collateral activity, which cleaves a reporter probe. In this study, we demonstrated that using 2-3 gRNAs in parallel can create a synergistic effect, resulting in a 4.5 × faster cleaving rate of the probe and increased sensitivity compared to using individual gRNAs. The synergy is due to the simultaneous activation of CRISPR-Cas12a and the improved performance of each gRNA. This approach was able to detect as few as 10 viral copies of the N-gene of SARS-CoV-2 RNA after a preamplification step using reverse transcription loop-mediated isothermal amplification. The method was able to accurately detect 100% of positive and negative clinical samples in â¼25 min using a fluorescence plate reader and â¼45 min with lateral flow strips.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/diagnóstico , Sistemas CRISPR-Cas/genética , RNA Viral/genética , Edição de Genes , RNA Guia de Sistemas CRISPR-CasRESUMO
Advances in the knowledge of the neuroendocrine system are closely related to the development of cellular imaging and labeling techniques. This synergy ranges from the staining techniques that allowed the first characterizations of the anterior pituitary gland, its relationship with the hypothalamus, and the birth of neuroendocrinology; through the development of fluorescence microscopy applications, specific labeling strategies, transgenic systems, and intracellular calcium sensors that enabled the study of processes and dynamics at the cellular and tissue level; until the advent of super-resolution microscopy, miniscopes, optogenetics, fiber photometry, and other imaging methods that allowed high spatiotemporal resolution and long-term three-dimensional cellular activity recordings in living systems in a conscious and freely moving condition. In this review, we briefly summarize the main contributions of cellular imaging techniques that have allowed relevant advances in the field of neuroendocrinology and paradigm shifts that have improved our understanding of the function of the hypothalamic-pituitary axes. The development of these methods and equipment is the result of the integration of knowledge achieved by the integration of several disciplines and effort to solve scientific questions and problems of high impact on health and society that this system entails.
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Hipotálamo , Neuroendocrinologia , Sistemas Neurossecretores , Diagnóstico por ImagemRESUMO
In spite of a current increasing trend in the development of miniaturized, standalone point-of-care (PoC) biosensing platforms in the literature, the actual implementation of such systems in the field is far from being a reality although deeply needed. In the particular case of the population screenings for local or regional diseases related to specific pathogens, the diagnosis of the presence of specific antibodies could drastically modify therapies and even the organization of public policies. The aim of this work was to develop a fast, cost-effective detection method based on the manipulation of functionalized magnetic beads for an efficient diagnosis of hypersensitivity pneumonitis (HP), looking for the presence of anti-pigeon antigen antibodies (APAA) in a patient's serum. We presented a Diagnostic Biosensor Method (DBM) in detail, with validation by comparison with a traditional high-throughput platform (ELISA assay). We also demonstrated that it was compatible with a microfluidic chip that could be eventually incorporated into a PoC for easy and broad deployment using portable optical detectors. After standardization of the different reaction steps, we constructed and validated a plastic chip that could easily be scaled to high-volume manufacturing in the future. The solution proved comparable to conventional ELISA assays traditionally performed by the clinicians in their laboratory and should be compatible with other antibody detection directly from patient samples.
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Alveolite Alérgica Extrínseca , Técnicas Biossensoriais , Alveolite Alérgica Extrínseca/diagnóstico , Anticorpos , Ensaio de Imunoadsorção Enzimática , Desenho de Equipamento , Humanos , Separação Imunomagnética , Dispositivos Lab-On-A-Chip , Microfluídica , Sistemas Automatizados de Assistência Junto ao LeitoRESUMO
The pituitary is a master endocrine gland that developed early in vertebrate evolution and therefore exists in all modern vertebrate classes. The last decade has transformed our view of this key organ. Traditionally, the pituitary has been viewed as a randomly organized collection of cells that respond to hypothalamic stimuli by secreting their content. However, recent studies have established that pituitary cells are organized in tightly wired large-scale networks that communicate with each other in both homo and heterotypic manners, allowing the gland to quickly adapt to changing physiological demands. These networks functionally decode and integrate the hypothalamic and systemic stimuli and serve to optimize the pituitary output into the generation of physiologically meaningful hormone pulses. The development of 3D imaging methods and transgenic models have allowed us to expand the research of functional pituitary networks into several vertebrate classes. Here we review the establishment of pituitary cell networks throughout vertebrate evolution and highlight the main perspectives and future directions needed to decipher the way by which pituitary networks serve to generate hormone pulses in vertebrates.
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Sistema Hipotálamo-Hipofisário/citologia , Sistema Hipotálamo-Hipofisário/metabolismo , Redes e Vias Metabólicas/fisiologia , Hipófise/citologia , Hipófise/metabolismo , Animais , Células Endócrinas/metabolismo , Gonadotrofos/metabolismo , Humanos , Hipotálamo/citologia , Hipotálamo/metabolismo , Filogenia , VertebradosRESUMO
Bilateral eye enucleation at birth (BE) leads to an expansion of the primary somatosensory cortex (S1) in rat pups. Although increased growth of the somatosensory thalamo-cortical afferents (STCAs) in part explains S1 expansion, timing mechanisms governing S1 formation are also involved. In this work, we begin the search of a developmental clock by intending to document the existence of putative clock neurons in the somatosensory thalamus (VPM) and S1 based upon changes of spontaneous spike amplitude; a biophysical property sensitive to circadian regulation; the latter known to be shifted by enucleation. In addition, we also evaluated whether STCAs growth rate and segregation timing were modified, as parameters the clock might time. We found that spontaneous spike amplitude transiently, but significantly, increased or decreased in VPM and S1 neurons of BE rat pups, respectively, as compared to their control counterparts. The growth rate and segregation timing of STCAs was, however, unaffected by BE. These results support the existence of a developmental clock that ticks differently in the VPM and S1 after BE. This observation, together with the fact that STCAs growth rate and segregation timing is unchanged, suggests that S1 expansion in BE rats may in part be controlled at the cortical level.
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Microfluidics has become a very promising technology in recent years, due to its great potential to revolutionize life-science solutions. Generic microfabrication processes have been progressively made available to academic laboratories thanks to cost-effective soft-lithography techniques and enabled important progress in applications like lab-on-chip platforms using rapid- prototyping. However, micron-sized features are required in most designs, especially in biomimetic cell culture platforms, imposing elevated costs of production associated with lithography and limiting the use of such devices. In most cases, however, only a small portion of the structures require high-resolution and cost may be decreased. In this work, we present a replica-molding method separating the fabrication steps of low (macro) and high (micro) resolutions and then merging the two scales in a single chip. The method consists of fabricating the largest possible area in inexpensive macromolds using simple techniques such as plastics micromilling, laser microfabrication, or even by shrinking printed polystyrene sheets. The microfeatures were made on a separated mold or onto existing macromolds using photolithography or 2-photon lithography. By limiting the expensive area to the essential, the time and cost of fabrication can be reduced. Polydimethylsiloxane (PDMS) microfluidic chips were successfully fabricated from the constructed molds and tested to validate our micro-macro method.
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Estrus synchronization induces cows to gather in sexually active groups (SAGs) composed of females displaying mounting activity. Although this technique promotes the enhancement of sexual behavior, there are cows in estrus (CE) that delay estrus expression and also cows not displaying estrus (CNDE) even in the presence of a preovulatory follicle (PF). To elucidate the physiological mechanisms of the delay in the onset of estrus or absence of estrus behavior, an observational study was undertaken in 17 Bos indicus cows treated with exogenous progesterone (CIDR) to synchronize estrus and to monitor follicular growth and its steroidogenic activity. After SAGs formation, cows were ovariectomized at 24, 48, and 72 h post-CIDR. Among ovariectomized groups there were only 9 CE which: 1) showed differences in the onset of estrus; 2) displayed distinctive follicular growth patterns; and 3) at 72 h produced the highest intrafollicular estradiol concentration, and showed a linear trend to increase expression of P450scc and P450arom. Comparison of CE vs. CNDE showed that: 1) both groups had progesterone levels indicative of cyclic activity, and a PF which grew at a similar rate and size; 2) CE showed a stronger association between time and growth; and 3) CE produced more intrafollicular estradiol and progesterone, together with the expression of higher levels of P450arom. Results suggest that pending on the pattern of growth of the PF and its steroidogenic potential to produce estradiol, the onset and expression of estrus behavior may be delayed probably until the establishment of the appropriate conditions to ensure ovulation.
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Estro/fisiologia , Fase Folicular/fisiologia , Fase Folicular/psicologia , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/metabolismo , Comportamento Sexual Animal/fisiologia , Animais , Aromatase/metabolismo , Bovinos , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Estradiol/metabolismo , Estro/sangue , Sincronização do Estro/métodos , Feminino , Fase Folicular/sangue , Folículo Ovariano/diagnóstico por imagem , Ovariectomia , Progesterona/metabolismo , Progesterona/farmacologia , Fatores de Tempo , UltrassonografiaRESUMO
Insulin secretion in mature beta-cells increases vigorously when extracellular glucose concentration rises. Glucose-stimulated insulin secretion depends on Ca(2+) influx through voltage-gated Ca(2+) channels. During fetal development, this structured response is not well established, and it is after birth that beta-cells acquire glucose sensitivity and a robust secretion. We compared some elements of glucose-induced insulin secretion coupling in beta-cells obtained from neonatal and adult rats and found that neonatal cells are functionally immature compared with adult cells. We observed that neonatal cells secrete less insulin and cannot sense changes in extracellular glucose concentrations. This could be partially explained because in neonates Ca(2+) current density and synthesis of mRNA alpha1 subunit Ca(2+) channel are lower than in adult cells. Interestingly, immunostaining for alpha1B, alpha1C, and alpha1D subunits in neonatal cells is similar in cytoplasm and plasma membrane, whereas it occurs predominantly in the plasma membrane in adult cells. We also observed that GLUT2 expression in adult beta-cells is mostly located in the membrane, whereas in neonatal cells glucose transporters are predominantly in the cytoplasm. This could explain, in part, the insensitivity to extracellular glucose in neonatal beta-cells. Understanding neonatal beta-cell physiology and maturation contributes toward a better comprehension of type 2 diabetes physiopathology, where alterations in beta-cells include diminished L-type Ca(2+) channels and GLUT2 expression that results in an insufficient insulin secretion.
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Envelhecimento/metabolismo , Canais de Cálcio/metabolismo , Transportador de Glucose Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Animais , Animais Recém-Nascidos , Canais de Cálcio/genética , Canais de Cálcio/fisiologia , Células Cultivadas , Condutividade Elétrica , Técnicas Imunológicas , Insulina/biossíntese , Insulina/genética , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Masculino , Isoformas de Proteínas/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Coloração e RotulagemRESUMO
Neonatal beta cells are functionally immature as they secrete less insulin than adults and lack of glucose response. The mechanisms that participate in the functional maturation of these cells are not known. Adult rat beta cells synthesize and secrete nerve growth factor (NGF) and express NGF receptors. NGF increases glucose-induced insulin secretion by modulating electrical activity in adult beta cells. In this work, we explored if NGF is involved in the maturation of glucose-induced insulin secretion coupling in rat neonate beta-cells.
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Canais de Cálcio/metabolismo , Glucose/metabolismo , Células Secretoras de Insulina/citologia , Insulina/metabolismo , Fator de Crescimento Neural/metabolismo , Animais , Animais Recém-Nascidos , Transporte Biológico , Fenômenos Fisiológicos Celulares , Secreção de Insulina , Masculino , Modelos Biológicos , RNA Mensageiro/metabolismo , RatosRESUMO
Numerous sperm functions including the acrosome reaction (AR) are associated with Ca(2+) influx through voltage-gated Ca(2+) (Ca(V)) channels. Although the electrophysiological characterization of Ca(2+) currents in mature sperm has proven difficult, functional studies have revealed the presence of low-threshold (Ca(V)3) channels in spermatogenic cells. However, the molecular identity of these proteins remains undefined. Here, we identified by reverse transcription polymerase chain reaction the expression of Ca(V)3.3 mRNA in mouse male germ cells, an isoform not previously described in these cells. Immunoconfocal microscopy revealed the presence of the three Ca(V)3 channel isoforms in mouse spermatogenic cells. In mature mouse sperm only Ca(V)3.1 and Ca(V)3.2 were detected in the head, suggesting its participation in the AR. Ca(V)3.1 and Ca(V)3.3 were found in the principal and the midpiece of the flagella. All Ca(V)3 channels are also present in human sperm, but only to a minor extent in the head. These findings were corroborated by immunogold transmission electron microscopy. Tail localization of Ca(V)3 channels suggested they may participate in motility, however, mibefradil and gossypol concentrations that inhibit Ca(V)3 channels did not significantly affect human sperm motility. Only higher mibefradil doses that can block high-threshold (HVA) Ca(V) channels caused small but significant motility alterations. Antibodies to HVA channels detected Ca(V)1.3 and Ca(V)2.3 in human sperm flagella.