Novel insights into the nervous system affected by prolonged hyperglycemia.

Multiple molecular pathways including the receptor for advanced glycation end-products-diaphanous related formin 1 (RAGE-Diaph1) signaling are known to play a role in diabetic peripheral neuropathy (DPN). Evidence suggests that neuropathological alterations in type 1 diabetic spinal cord may occur at the same time as or following peripheral nerve abnormalities. We demonstrated that DPN was associated with perturbations of RAGE-Diaph1 signaling pathway in peripheral nerve accompanied by widespread spinal cord molecular changes. More than 500 differentially expressed genes (DEGs) belonging to multiple functional pathways were identified in diabetic spinal cord and of those the most enriched was RAGE-Diaph1 related PI3K-Akt pathway. Only seven of spinal cord DEGs overlapped with DEGs from type 1 diabetic sciatic nerve and only a single gene cathepsin E (CTSE) was common for both type 1 and type 2 diabetic mice. In silico analysis suggests that molecular changes in spinal cord may act synergistically with RAGE-Diaph1 signaling axis in the peripheral nerve. KEY MESSAGES: Molecular perturbations in spinal cord may be involved in the progression of diabetic peripheral neuropathy. Diabetic peripheral neuropathy was associated with perturbations of RAGE-Diaph1 signaling pathway in peripheral nerve accompanied by widespread spinal cord molecular changes. In silico analysis revealed that PI3K-Akt signaling axis related to RAGE-Diaph1 was the most enriched biological pathway in diabetic spinal cord. Cathepsin E may be the target molecular hub for intervention against diabetic peripheral neuropathy.

Isolation of Citrus lemon extracellular vesicles: Development and process control using capillary electrophoresis.

A new and scalable method for the isolation of extracellular vesicles (EV) from Citrus lemon juice samples was developed. The methodology included preliminary preconcentration of the sample using ultrafiltration (UF) followed by size-exclusion chromatography (SEC) purification and final preconcentration of the eluates. Transmission electron microscopy and proteomic analysis showed that isolates contained exosome-like vesicles, exocyst-positive organelle (EXPO), and microvesicles. The efficiency of certain isolation steps was evaluated with total protein content assay (bicinchoninic acid assay, BCA), nanoparticles tracking analysis (NTA), and capillary electrophoresis (CE). A good correlation between CE, BCA, and NTA results was shown. The application of CE enabled the detection of soluble contaminants, macromolecular aggregates, and vesicles' heterogeneity. The fluorescent staining of encapsulated nucleic acids was proposed for the identity confirmation of EV detected in CE. The study demonstrates the CE as a comprehensive tool for monitoring of the EV isolation process.

Low-Intensity Blue Light Exposure Reduces Melanopsin Expression in Intrinsically Photosensitive Retinal Ganglion Cells and Damages Mitochondria in Retinal Ganglion Cells in Wistar Rats.

This study investigated the effect of low-intensity blue light on the albino Wistar rat retina, including intrinsically photosensitive retinal ganglion cells (ipRGCs). Three groups of nine albino Wistar rats were used. One group was continuously exposed to blue light (150 lx) for 2 d (STE); one was exposed to 12 h of blue light and 12 h of darkness for 10 d (LTE); one was maintained in 12 h of white light (150 lx) and 12 h of darkness for 10 d (control). Melanopsin (Opn4) was immunolabelled on retinal whole-mounts. To count and measure Opn4-positive ipRGC somas and dendrites (including Sholl profiles), Neuron J was used. Retinal cryosections were immunolabeled for glial fibrillary acid protein (GFAP) and with terminal deoxynucleotidyl transferase dUTP nick-end labelling for apoptosis detection. LTE reduced the length of Opn4-positive ipRGC dendrites (p = 0.03) and decreased Opn4-immunoreactivity in ipRGC outer stratifying dendrites. LTE and STE decreased the complexity of dendritic arborization (Sholl profile; p < 0.001, p = 0.03, respectively), increased retinal GFAP immunoreactivity (p < 0.001, p = 0.002, respectively), and caused outer segment vesiculation and outer nuclear layer apoptosis. Ultrastructural analysis showed that LTE damaged mitochondria in retinal ganglion cells and in the inner plexiform layer. Thus, LTE to low-intensity blue light harms the retinas of albino Wistar rats.

Histology and Ultrastructure of the Esophagus in European Beaver (Castor fiber) Displays Features Adapted to Seasonal Changes in Diet.

The European beaver is a herbivorous rodent whose diet changes seasonally, and in winter consists of large quantities of woody plants. It is distinguished among other mammals by a unique organization of the stomach that comprises the cardiogastric gland and by the unusual process of mucus formation in the gastric mucosa. The aim of study was to (i) characterize the structure of the beaver esophagus with particular attention to the mucosal epithelium; (ii) compare the histological structure of the esophagi collected in spring, summer, and winter; (iii) provide preliminary data on the structure of the esophagus in beaver fetuses. The study was conducted on esophagi of 18 adult beavers captured in Poland in April, August, and December, and on 3 fetal organs. The results obtained in adults show that the mucosa is lined with thick stratified squamous keratinized epithelium with a structure similar to that of the skin epidermis. Ultrastructural studies reveal the presence of multiple lamellar and non-lamellar bodies in granular cells, whose morphology and location gradually change while reaching the upper epithelial layers. The muscularis mucosa comprises a layer of longitudinally oriented bundles of smooth muscle cells. Both mucosa and submucosa do not comprise any glands. The thick muscularis externa consists mainly of internal circular and external longitudinal layers of striated muscle fibers. The keratinized layer of mucosa epithelium was 2-3-fold thicker in esophagi collected in winter than in those collected in spring and summer, while the epithelial cell layer thickness remained unchanged regardless of the season. Immunolabeling for proliferating cell nuclear antigen shows a higher index of epithelium proliferation in esophagi collected in winter than in spring and summer. No seasonal differences were noted in other layers of the esophagus. Fetal organs have epithelium covered with a keratinized layer, thinner than in adults, and the muscularis externa comprises both striated and smooth muscle cells.

The Use of Chitin from the Molts of Mealworm (Tenebrio molitor) for the Removal of Anionic and Cationic Dyes from Aqueous Solutions.

The possibility of using chitin from the molts of an insect-ealworm (Tenebrio molitor) to remove anionic (RB5, RY84) and cationic dyes (BV10, BR46) from aqueous solutions was investigated. The scope of the research included, among others: Characteristics of chitin from mealworms (FTIR, SEM, pHPZC), the effect of pH on sorption efficiency, sorption kinetics (pseudo-first, pseudo-second order, intramolecular diffusion models) and the determination of the maximum sorption capacity (Langmuir and Freundlich models). The sorption efficiency of anionic dyes on chitin from mealworm was the highest at pH 2-3, and for cationic dyes at pH 6. The equilibrium time of sorption of anionic dyes was 240-300 min and for cationic dyes it was 180-240 min. The experimental data on dye sorption kinetics was best described by the pseudo-second order model. The maximum sorption capacity of chitin from the mealworm for the anionic dyes RB5 and RY84 was 121.15 mg/g and 138.55 mg/g, respectively, and was higher than with some carbon-based materials (literature data). In the case of cationic dyes, the sorption capacity of the tested chitin was lower and reached 3.22 mg/g and 59.56 mg/g for BV10 and BR46, respectively.

Profiles of Rho, Opn4, c-Fos, and Birc5 mRNA expression in Wistar rat retinas exposed to white or monochromatic light.

Despite concern over potential retinal damage linked to exposure to light-emitting-diode (LED) light (particularly blue light), it remains unknown how exposure to low-intensity monochromatic LED light affects the expression of rhodopsin (Rho, a photopigment that mediates light-induced retinal degeneration), melanopsin (Opn4, a blue-light sensitive photopigment), c-Fos (associated with retinal damage/degeneration), and Birc5 (anti-apoptotic). This study investigated the mRNA expression profiles of these genes under exposure to white and monochromatic light (blue, red, green) in the retinas of albino rats under a cycle of 12 h of light and 12 h of darkness. In each group, 32 Wistar rats were exposed to one type of monochromatic-LED or white-fluorescent light for 7 day (150 lx). Retinal samples were taken for qPCR analysis and light and electron microscopy. Blue and green light exposure markedly decreased expression of Rho and Opn4 mRNA and increased expression of Birc5 and c-Fos mRNA (P < 0.05). In retinas from the blue-light group, loss and vesiculation of photoreceptor outer segments were visible, but not in retinas from the red-light and control group. Measurements of the photoreceptor inner and outer segments length revealed, that this length was significantly decreased in the blue- and green-light exposure groups (P < 0.02), but not in the red-light exposure group. Increased expression of Birc5 and decreased expression of Rho and Opn4 after exposure to blue and green light may be early responses that help to reduce light-induced retinal damage.

Changes in the Metabolic Profile of Melatonin Synthesis-Related Indoles during Post-Embryonic Development of the Turkey Pineal Organ.

Research on age-dependent changes in pineal activity has been limited almost exclusively to melatonin (MLT). This study determined, for the first time, the alterations occurring in the metabolic profile of MLT synthesis-related indoles during the post-embryonic development period in birds. Turkeys reared under a 12 h light/dark cycle were euthanized at 2 h intervals for 24 h at the ages of 2, 7, 14, and 28 days and 10, 20, 30, and 45 weeks. The results showed prominent changes in the metabolic profile of indoles during development and could be distinguished in four stages. The first stage, from hatching to the age of 2 weeks, was characterized by a decrease in the 5-hydroxytryptophan concentration and an increase in the concentrations of serotonin (5-HT), MLT, 5-methoxyindoleacetic acid, and 5-methoxytryptamine (5-MTAM). During the second stage, around the age of 1 month, the concentrations of N-acetylserotonin (NAS) and MLT reached a maximum. The synthesis and degradation of 5-HT were also the highest. The third stage, around the age of 10 weeks, was characterized by decreased levels of 5-HT (approximately 50%) and 5-hydroxyindoleacetic acid and a high level of 5-MTAM. The last stage, covering the age of 20 to 45 weeks, was characterized by a large decrease in the synthesis, content, and degradation of 5-HT. Despite these changes, there were no prominent differences in the nocturnal levels of NAS and MLT between the third and fourth stages. The concentrations of all tryptophan derivatives showed daily fluctuations until the age of 45 weeks.

Field-Emission Scanning Electron Microscope as a Tool for Large-Area and Large-Volume Ultrastructural Studies.

The development of field-emission scanning electron microscopes for high-resolution imaging at very low acceleration voltages and equipped with highly sensitive detectors of backscattered electrons (BSE) has enabled transmission electron microscopy (TEM)-like imaging of the cut surfaces of tissue blocks, which are impermeable to the electron beam, or tissue sections mounted on the solid substrates. This has resulted in the development of methods that simplify and accelerate ultrastructural studies of large areas and volumes of biological samples. This article provides an overview of these methods, including their advantages and disadvantages. The imaging of large sample areas can be performed using two methods based on the detection of transmitted electrons or BSE. Effective imaging using BSE requires special fixation and en bloc contrasting of samples. BSE imaging has resulted in the development of volume imaging techniques, including array tomography (AT) and serial block-face imaging (SBF-SEM). In AT, serial ultrathin sections are collected manually on a solid substrate such as a glass and silicon wafer or automatically on a tape using a special ultramicrotome. The imaging of serial sections is used to obtain three-dimensional (3D) information. SBF-SEM is based on removing the top layer of a resin-embedded sample using an ultramicrotome inside the SEM specimen chamber and then imaging the exposed surface with a BSE detector. The steps of cutting and imaging the resin block are repeated hundreds or thousands of times to obtain a z-stack for 3D analyses.

Embryonic Development of Avian Pineal Secretory Activity-A Lesson from the Goose Pineal Organs in Superfusion Culture.

The embryonic ontogeny of pineal secretory activity in birds has been investigated almost exclusively in chickens. This study aimed to characterize this process in domestic geese. The pineal organs of embryos aged 18-28 days were incubated in superfusion culture under different light conditions for 4-5 days and treated with norepinephrine (NE). Melatonin (MLT) was measured by radioimmunoassay and other indoles by HPLC with fluorescence detection. Additionally, pineal organs were collected from embryos at 14-28 days of age and used to measure catecholamines by HPLC with electrochemical detection. MLT secretion increased with embryo age, most intensively between the 22nd and 24th days of life. The daily changes in MLT secretion under the 12 L:12D cycle occurred on the first day of culture, starting from an embryonic age of 24 days. MLT secretion was controlled by the light-dark cycle in all age groups studied. However, exposure to light during the scotophase did not alter the secretion of MLT. The endogenous oscillator expressed its activity in regulating MLT secretion in the pineal organs of embryos aged 24 days and older but could not generate a rhythm after one cycle. The rhythm of 5-hydroxytryptophan release during the first day of culture was found in the pineal organs of all embryos, while the rhythmic release of N-acetylserotonin and 5-methoxyindole acetic acid started at the age of 24 days. The proportion of released indoles changed with embryo age. NE caused a decrease in MLT secretion and provoked an increase in serotonin release. Incubation of the pineal organs induced the development of MLT secretory machinery and its diurnal rhythmicity. The pineal content of catecholamines increased prominently at the end of embryonic development.

Norepinephrine Is a Major Regulator of Pineal Gland Secretory Activity in the Domestic Goose (Anser anser).

This study determined the effect of norepinephrine and light exposure on melatonin secretion in goose pineal explants. Additionally, it investigated changes in the content of norepinephrine, dopamine, and their metabolites [3,4-dihydroxyphenylacetic acid; vanillylmandelic acid (VMA); homovanillic acid] in goose pineal glands in vivo under 12 h of light and 12 h of darkness (LD), a reversed cycle (DL), constant light (LL), and constant darkness (DD). In vitro content of melatonin was measured by radioimmunoassay; contents of catecholamines and their metabolites were measured by high-performance liquid chromatography. Exposure of pineal explants to LD or DL established rhythmic melatonin secretion; this rhythm was much better entrained with norepinephrine exposure during photophase than without it. When the explants were kept in LL or DD, the rhythm was abolished, unless NE was administered during natural scotophase of a daily cycle. In vivo, norepinephrine and dopamine levels did not display rhythmic changes, but their respective metabolites, HMV and VMA, displayed well-entrained diurnal rhythms. These results indicate that norepinephrine and sympathetic innervation play key roles in regulation of pineal secretory activity in geese, and that pineal levels of VMA and HMV provide precise information about the activity of sympathetic nerve fibers in goose pineal glands.

Metabolism of Melatonin Synthesis-Related Indoles in the Turkey Pineal Organ and Its Modification by Monochromatic Light.

The metabolism of pineal indoles is closely related to alterations in the light and dark phases of a daily cycle. Recent research showed important interspecies differences in the pineal biochemistry, and a strong impact of monochromatic light on many physiological processes in birds. Therefore, the aims of study were to characterize the metabolism of melatonin-synthesis indoles in the pineal organ of the domestic turkey, and to determine the changes occurring in this metabolism under the influence of different wavelengths and intensities of light. For this purpose, 3-week-old turkeys were kept under 16 lx white light, or under blue, green, and red light with intensities of 16, 32, and 64 lx during the photophase, and after 7 d were sacrificed at 4 h intervals. The activities of melatonin-synthesizing enzymes and the contents of indoles were measured in the same pineal organ. The results revealed that the activities of tryptophan hydroxylase and arylalkylamine N-acetyltransferase, and the levels of all tryptophan derivatives had significant daily changes in birds kept under each light condition used. The profile of pineal indole metabolism in 4-week-old turkeys was characterized by high-amplitude rhythms in the activity of arylalkylamine N-acetyltransferase and the contents of N-acetylserotonin and melatonin, equal relative amounts of serotonin and 5-hydroxyindoleacetic acid, and higher content of melatonin than N-acetylserotonin. The monochromatic light significantly modified the pineal indole metabolism, and its effects were dependent on the color and intensity of light. Pronounced changes occurred in the level of serotonin synthesis and the daily rhythm course of melatonin synthesis.

Effects of Deoxynivalenol and Zearalenone on the Histology and Ultrastructure of Pig Liver.

The purpose of this study was to determine the effects of single and combined administrations of deoxynivalenol (DON) and zearalenone (ZEN) on the histology and ultrastructure of pig liver. The study was performed on immature gilts, which were divided into four equal groups. Animals in the experimental groups received DON at a dose of 12 µg/kg body weight (BW) per day, ZEN at 40 µg/kg BW per day, or a mixture of DON (12 µg/kg BW per day) and ZEN (40 µg/kg BW). The control group received vehicle. The animals were killed after 1, 3, and 6 weeks of experiment. Treatment with mycotoxins resulted in several changes in liver histology and ultrastructure, including: (1) an increase in the thickness of the perilobular connective tissue and its penetration to the lobules in gilts receiving DON and DON + ZEN; (2) an increase in the total microscopic liver score (histology activity index (HAI)) in pigs receiving DON and DON + ZEN; (3) dilatation of hepatic sinusoids in pigs receiving ZEN, DON and DON + ZEN; (4) temporary changes in glycogen content in all experimental groups; (5) an increase in iron accumulation in the hepatocytes of gilts treated with ZEN and DON + ZEN; (6) changes in endoplasmic reticulum organization in the hepatocytes of pigs receiving toxins; (7) changes in morphology of Browicz-Kupffer cells after treatment with ZEN, DON, and DON + ZEN. The results show that low doses of mycotoxins used in the present study, even when applied for a short period, affected liver morphology.

Diurnal Rhythm of Plasma Melatonin Concentration in the Domestic Turkey and Its Regulation by Light and Endogenous Oscillators.

The aim of this study was to characterize the diurnal rhythm of plasma melatonin (MLT) concentration and its regulation by light and endogenous oscillators in 10-week-old domestic turkeys. Three experiments were performed to examine (i) the course of daily changes in plasma MLT concentration in turkeys kept under a 12 h light: 12 h dark (12L:12D) cycle; (ii) the influence of night-time light exposure lasting 0.5, 1, 2, or 3 h on the plasma MLT level; and (iii) the occurrence of circadian fluctuations in plasma MLT levels in birds kept under continuous dim red light and the ability of turkeys to adapt their pineal secretory activity to a reversed light-dark cycle (12D:12L). The plasma MLT concentration was measured with a direct radioimmunoassay. The plasma MLT concentration in turkeys kept under a 12L:12D cycle changed significantly in a daily rhythm. It was low during the photophase and increased stepwise after the onset of darkness to achieve the maximal level in the middle of the scotophase. Next, it decreased during the second half of the night. The difference between the lowest level of MLT and the highest level was approximately 18-fold. The exposure of turkeys to light during the scotophase caused a rapid, large decrease in plasma MLT concentration. The plasma MLT concentration decreased approximately 3- and 10-fold after 0.5 and 1 h of light exposure, respectively, and reached the day-time level after 2 h of exposure. In turkeys kept under continuous darkness, the plasma MLT level was approximately 2.5-fold higher at 02:00 h than at 14:00 h. In birds kept under 12D:12L, the plasma MLT level was significantly higher at 14:00 h than at 02:00 h. The results showed that plasma MLT concentrations in 10-week-old turkeys have a prominent diurnal rhythm, which is endogenously generated and strongly influenced by environmental light.

Capillary zone electrophoresis of bacterial extracellular vesicles: A proof of concept.

The extracellular vesicles (EVs) released by plant pathogens of the Pectobacterium genus were investigated. The isolates were obtained using differential centrifugation followed by filtration and were characterized in terms of total protein content and particle size distribution. The transmission electron microscopy (TEM) analysis revealed the presence of two morphologically differentiated subpopulations of vesicles in the obtained isolates. The proteomic analysis using matrix-assisted laser desorption ionization mass spectrometry with time of flight detector (MALDI-TOF/TOF-MS) enabled to identify 62 proteomic markers commonly found in EVs of Gram-negative rods from the Enterobacteriaceae family. Capillary electrophoresis (CE) was proposed as a novel tool for the characterization of EVs. The method allowed for automated and fast (<15 min per sample) separation of vesicles from macromolecular aggregates with low sample consumption (about 10 nL per analysis). The approach required simple background electrolyte (BGE) composed of 50 mM BTP and 75 mM glycine (pH 9.5) and standard UV detection. The report presents a new opportunity for quality control of samples containing EVs.

Pinopsin and photoreception in the pineal organ of the domestic turkey during post-hatching development.

The avian pineal organ is photosensitive because of the presence of photopigments, of which pinopsin seems to be one of the most important. This organ is subject to far-reaching changes during post-hatching development, but evidence regarding pinopsin presence and direct photoreception during this time is lacking. This study was carried out to demonstrate the following: 1) the structures showing immunoreactivity to pinopsin in the turkey pineal organ, 2) the changes of these structures during development, 3) the pinopsin localization in pinealocytes in monolayer cultures, and 4) the role of direct photoreception in the regulation of melatonin secretion in pineal organs in adult turkeys. Pinopsin immunoreactivity was localized in the apical extensions of columnar cells limiting the follicular lumen, in fiber-like structures located between columnar cells in the inner part of follicle wall, in string-shapes or small spherical structures distributed in the outer part of follicle wall and in amorphous material inside the follicle lumen. In young birds, immunoreactivity was also sporadically noted in cell bodies of rudimentary receptor pinealocytes. The distribution of pinopsin showed prominent age-dependent changes, including a subsequent increase in pinopsin-positive structures in the outer part of the follicle wall and a prominent reduction in the number and size of positive apical extensions in 40- and 56-week-old turkeys. These data demonstrate that the role of secretory pinealocytes in pineal photoreception increases with age. In monolayer cultures, all pinealocytes showed strong reactions in club- or bulbous-shaped prolongations. The pineal organs of adult birds were less sensitive to light exposition at night than those of young turkeys, which points to differences in light sensitivity between rudimentary receptor and secretory pinealocytes. However, direct photoreception could play an important role in the regulation of melatonin secretion in adult turkeys.

Diurnal and circadian variations in intraocular pressure in goats exposed to different lighting conditions.

The effect of constant light and constant darkness on intraocular pressure (IOP) in goats has not been investigated. We hypothesized that IOP variations would differ between goats kept under a cycle of 12 hours of light and 12 hours of darkness (LD), constant darkness (DD), and constant light (LL). To test this hypothesis, goats were exposed to these conditions for five days (LD, 30 goats; DD, 10 goats; LL, 10 goats). IOP was measured by applanation tonometry at 9 a.m. (beginning of photophase in LD) and 9 p.m. (beginning of scotophase in LD) on the fourth and fifth days of exposure. We found that changes in mean IOP from 9 a.m. to 9 p.m. differed significantly between groups (χ2(2) = 23.04, p < .0001). Most goats in LD showed a regular pattern of higher IOP in the morning and lower IOP in the evening, whereas those in DD and LL did not follow this pattern. In LD conditions, mean IOP was 2.4 mm Hg lower at 9 p.m. than at 9 a.m. (95% confidence interval for the difference (CI): -2.8 to -1.9 mm Hg, p < .0001). In DD conditions, mean IOP did not differ between 9 p.m. and 9 a.m. (CI: -0.9 to 0.8 mm Hg, p = .90). In LL conditions, it was 0.6 mm Hg lower at 9 p.m. (CI: -1.5 to 0.2 mm Hg, p = .12). Our results indicate that IOP in goats kept in LD is higher in the morning than in the evening, and that IOP variations are reduced in goats kept in DD and LL. These results suggest that exposure to alternating periods of light and darkness is important for maintaining rhythmic variations in IOP in this species.

Roles of Direct Photoreception and the Internal Circadian Oscillator in the Regulation of Melatonin Secretion in the Pineal Organ of the Domestic Turkey: A Novel In Vitro Clock and Calendar Model.

The regulation of melatonin secretion in the avian pineal organ is highly complex and shows prominent interspecies differences. The aim of this study was to determine the roles of direct photoreception and the internal oscillator in the regulation of melatonin secretion in the pineal organ of the domestic turkey. The pineal organs were collected from 12-, 13- and 14-week-old female turkeys reared under a 12 L:12 D cycle with the photophase from 07.00 to 19.00, and were incubated in superfusion culture for 3-6 days. The cultures were subjected to different light conditions including 12 L:12 D cycles with photophases between 07.00 and 19.00, 13.00 and 01.00 or 01.00 and 13.00, a reversed cycle 12 D:12 L, cycles with long (16 L:8 D) and short (8 L:16 D) photophases, and continuous darkness or illumination. The pineal organs were also exposed to light pulses of variable duration during incubation in darkness or to periods of darkness during the photophase. The secretion of melatonin was determined by direct radioimmunoassay. The turkey pineal organs secreted melatonin in a well-entrained diurnal rhythm with a very high amplitude. Direct photoreception as an independently acting mechanism was able to ensure quick and precise adaptation of the melatonin secretion rhythm to changes in light-dark conditions. The pineal organs secreted melatonin in circadian rhythms during incubation in continuous darkness or illumination. The endogenous oscillator of turkey pinealocytes was able to acquire and store information about the light-dark cycle and then to generate the circadian rhythm of melatonin secretion in continuous darkness according to the stored data. The obtained data suggest that the turkey pineal gland is highly autonomous in the generation and regulation of the melatonin secretion rhythm. They also demonstrate that the turkey pineal organ in superfusion culture is a valuable model for chronobiological studies, providing a highly precise clock and calendar. This system has several features which make it an attractive alternative to other avian pineal glands for circadian studies.

Embryonic Ontogeny of 5-Hydroxyindoles and 5-Methoxyindoles Synthesis Pathways in the Goose Pineal Organ.

The aim of this study was to characterize the embryonic ontogeny of 5-hydroxyindoles and 5-methoxyindoles synthesis pathways in the goose pineal organ. The study was performed on embryos aged 14-28 days, which have been incubated under a 12L:12D cycle. The pineal organs were collected for measurements of indole content by HPLC every 6 h on embryonic day (ED) 14, ED 16, ED 18 and ED 22 or every 2 h on ED 24, ED 26 and ED 28. The level of tryptophan showed no significant changes during development and no day-night variations. The content of 5-hydroxytryptophan increased between ED 14 and ED 26. It was significantly higher during scotophase than during photophase starting from ED 14. The serotonin content was low during the early stages of development (ED 14-ED 18) and prominently increased from ED 20. The serotonin levels also showed day-night differences; however, they were less conspicuous than those of 5-hydroxytryptophan. The changes in the level of 5-hydroxyindole acetic acid were similar to those of serotonin. 5-Hydroxytryptophol was measurable from ED 18. Levels of N-acetylserotonin, which were detectable for the first time on ED 16, prominently increased between ED 22 and ED 28 and showed significant day-night differences from ED 20. Melatonin was detectable from ED 18. Like N-acetylserotonin, its content increased rapidly between ED 22 and ED 28, and from ED 20 showed diurnal variations. 5-Methoxyindole acetic acid and 5-methoxytryptophol occurred at measurable levels from ED 18 and ED 26, respectively. The obtained results showed that embryonic development of indole metabolism in the goose pineal organ starts with the beginning of serotonin synthesis. The processes of serotonin acetylation and 5-hydroxyindoles methylation were turned on later. Diurnal rhythmicity develops very early in the embryonic pineal organ of the goose when the eggs are incubated under a 12 h light: 12 h dark schedule. Two processes are responsible for generation of the diurnal rhythms of 5-hydroxyindoles and 5-methoxyindoles: (i) hydroxylation of tryptophan and (ii) acetylation of serotonin.

Developmental morphology of the turkey pineal organ. Immunocytochemical and ultrastructural studies.

Our previous study showed that the turkey pineal organ, in contrast to that of the chicken, is characterized by a follicular structure throughout the entire period of post-hatching life. Despite the preservation of the follicular organization, the histological structure of the pineal follicles in turkeys changes prominently with age. The present research was performed to investigate the cellular composition and organization of the follicle wall as well as the ultrastructure of parenchymal cells in the turkey pineal organ during the period of post-hatching development. Pineal organs were collected from female turkeys at 2 days, 2 weeks, 4 weeks, 10 weeks, 20 weeks, 30 weeks, 40 weeks, and 56 weeks post-hatching. The organs were prepared for immunocytochemical studies using antibodies against N-acetylserotonin O-methyltransferase (ASMT), glial fibrillary acidic protein (GFAP) and proliferating cell nuclear antigen (PCNA) and for ultrastructural examination. The results showed that regardless of age, the pineal follicle was formed by ASMT-immunopositive cells, among which rudimentary photoreceptor and secretory pinealocytes were identified. The second component of the follicle wall consisted of GFAP-immunopositive cells, as represented by ependymal-like and astrocyte-like cells. Rudimentary photoreceptor pinealocytes and ependymal-like cells formed the inner part of the follicle wall, while secretory pinealocytes and astrocyte-like cells created the outer part. Three forms of the pineal follicle structure characteristic of young (two days to ten weeks), young adult (20-30 weeks) and adult (40-56 weeks) turkeys were distinguished. These forms primarily differed in the relative dimensions of the inner and outer parts of the follicle wall. Ultrastructural studies showed prominent changes in the organization of rudimentary receptor pinealocytes during the investigated period of life. These cells developed until the age of 20 weeks, at which time they appeared as strongly elongated cells with a stratified, highly regular distribution of organelles. In adult turkeys, rudimentary receptor pinealocytes showed pronounced regressive changes; however, we never observed their transformation into cells of the secretory type. Secretory pinealocytes increased in number and size during the post-hatching period, which was especially pronounced after 20 weeks of age. The most prominent changes in the supporting cells included the intensification of GFAP-immunoreactivity due to the accumulation of filaments in the cytoplasm and the development of astrocyte-like cells. The increase in the number of secretory pinealocytes and astrocyte-like supporting cells resulted in the formation of two distinct parts of the follicle wall in the pineal organs of young adult and adult turkeys.

In vivo miRNA delivery in whitefish: Synthetic MiR92b-3p uptake and the efficacy of gene expression silencing.

IMPACT STATEMENT: The delivery of short snippets of RNA, such as synthetic miRNA agents, is an essential step for achieving RNA-mediated knockdown, which has not been studied in sufficient detail in fish. Our results indicate that a MiR92b-3p mimic may be effectively delivered via intraperitoneal injection to the spleen and the liver of whitefish, and that it likely achieves functionality without causing any apparent toxic effects in the challenged animals. We report the novel finding that the MiR92b-3p mimic reduced the in vivo liver mRNA expression levels of its putative pro-apoptotic targets (p53, cdkn1a, and pcna), and important metabolic genes, e.g. cdo1. This shows that this methodology of MiR92b-3p mimic transfection in vivo may be a useful tool for studies that investigate the molecular pathways that confer pro-proliferative and anti-apoptotic phenotypes or those that regulate intracellular metabolism in fish and other vertebrates.