Search

There has been a growing interest in the use of hemp as an animal feed ingredient considering its economic value and nutritional properties. However, there is a paucity of research regarding the safety of hemp-based animal feed currently. Thus, this raises safety concerns on the potential transfer of cannabinoids from hemp-based animal feed to animal products intended for human consumption and its health effects. As such, the detection and quantification of cannabinoids in meat and animal feeds would be desirable for monitoring purposes. In this study, a simple, rapid and sensitive method for the simultaneous quantification of four major cannabinoids (delta-9-tetrahydrocannabinol, cannabidiol, cannabinol and tetrahydrocannabinolic acid) in meat and animal feeds by liquid chromatography–tandem mass spectrometry (LC-MS/MS) was successfully developed and validated. The method was selective and sensitive, achieving limits of detection and quantification for the four cannabinoids from 5 to 7 µg/kg and 15 to 21 µg/kg, respectively. The overall recovery with matrix-matched calibration curves for the cannabinoids ranged from 87–115%. The coefficients of variation were between 2.17–13.38% for intraday precision and 3.67–12.14% for inter-day precision. The method was subsequently applied to monitor cannabinoids in 120 meat and 24 animal feed samples. No cannabinoid was detected, suggesting no imminent food safety concerns arising from the potential incorporation of hemp and by-products in animal feed and nutrition under the promotion of sustainable agricultural practices. [ABSTRACT FROM AUTHOR]

The main functions of the mammalian cecum include microbial digestion of soluble and insoluble carbohydrates and the absorption of significant amounts of water and electrolytes. The present study was carried out to investigate the gross anatomy and electron microscopy of the cecum in adult camels (Camelus dromedarius), which still needs to be clarified. The study was conducted using samples collected from camels slaughtered at an abattoir in Omdurman, Sudan. The camel cecum, which occupied the right part of the abdomen, was situated caudally to the abomasum and directed caudally to the pelvis. It was short and S-shaped and began largely at the ceco-colic junction at the level of the fifth lumbar vertebra and became smaller towards its apex. Morphometrically, camel cecum constituted 3.2% of the large intestine length, 1% of both the small intestine and large intestine length, and 0.10% of the long axis of the animal body. Ultra-structurally, the cecal mucosa showed several folds and was lined by a simple columnar epithelium with many intestinal cells (enterocytes) and goblet cells; two types of enterocytes were shown: type one contained a dark cytoplasm and type two had a light cytoplasm. In conclusion, the cecum in dromedary camels was generally similar in structure to other domestic animals; however, they have a shorter cecum compared to ruminants.

There has been a growing interest in the use of hemp as an animal feed ingredient considering its economic value and nutritional properties. However, there is a paucity of research regarding the safety of hemp-based animal feed currently. Thus, this raises safety concerns on the potential transfer of cannabinoids from hemp-based animal feed to animal products intended for human consumption and its health effects. As such, the detection and quantification of cannabinoids in meat and animal feeds would be desirable for monitoring purposes. In this study, a simple, rapid and sensitive method for the simultaneous quantification of four major cannabinoids (delta-9-tetrahydrocannabinol, cannabidiol, cannabinol and tetrahydrocannabinolic acid) in meat and animal feeds by liquid chromatography–tandem mass spectrometry (LC-MS/MS) was successfully developed and validated. The method was selective and sensitive, achieving limits of detection and quantification for the four cannabinoids from 5 to 7 µg/kg and 15 to 21 µg/kg, respectively. The overall recovery with matrix-matched calibration curves for the cannabinoids ranged from 87–115%. The coefficients of variation were between 2.17–13.38% for intraday precision and 3.67–12.14% for inter-day precision. The method was subsequently applied to monitor cannabinoids in 120 meat and 24 animal feed samples. No cannabinoid was detected, suggesting no imminent food safety concerns arising from the potential incorporation of hemp and by-products in animal feed and nutrition under the promotion of sustainable agricultural practices.

The excessive accumulation of antibiotic residue in animal-derived foods pose a threat to our heath. Therefore, it is necessary to establish a rapid, simple, effective, safe and highly sensitive lincomycin (LIN) residues detection method by high-performance liquid chromatography equipped with ultraviolet. Here, due to the weak UV absorption of LIN, the p-Toluenesulfonyl isocyanate (PTSI, has a strong UV absorption) was binded to the hydroxyl group of LINs by using pre-column derivatization method, improving the UV absorption of LIN. This 9-min-run analytical method detects and quantifies LIN residues with acceptable validation performance parameters in terms of sensitivity, selectivity, linearity, the limit of quantification, accuracy, and precision.Our result showed that the limits of detection and quantification for LIN in samples were 25–40 μg/kg and 40–60 μg/kg, respectively, under the optimized conditions. Furthermore, the LIN recoveries ranged from 72.4 % to 96.7 %; the determination coefficients were 0.9912–0.9996, and the relative standard deviations were 5.6 %-12.7 %. Therefore, the HPLC-UVD method for detecting LIN residues in animal-derived food products has been established.

Brain microbleeds are increased in chronic kidney disease (CKD) and their presence increases risk of cognitive decline and stroke. We examined the interaction between CKD and brain microhemorrhages (the neuropathological substrate of microbleeds) in mouse and cell culture models and studied progression of microbleed burden on serial brain imaging from humans. Mouse studies: Two CKD models were investigated: adenine-induced tubulointerstitial nephritis and surgical 5/6 nephrectomy. Cell culture studies: bEnd.3 mouse brain endothelial cells were grown to confluence, and monolayer integrity was measured after exposure to 5-15% human uremic serum or increasing concentrations of urea. Human studies: Progression of brain microbleeds was evaluated on serial MRI from control, pre-dialysis CKD, and dialysis patients. Microhemorrhages were increased 2-2.5-fold in mice with CKD independent of higher blood pressure in the 5/6 nephrectomy model. IgG staining was increased in CKD animals, consistent with increased blood-brain barrier permeability. Incubation of bEnd.3 cells with uremic serum or elevated urea produced a dose-dependent drop in trans-endothelial electrical resistance. Elevated urea induced actin cytoskeleton derangements and decreased claudin-5 expression. In human subjects, prevalence of microbleeds was 50% in both CKD cohorts compared with 10% in age-matched controls. More patients in the dialysis cohort had increased microbleeds on follow-up MRI after 1.5 years. CKD disrupts the blood-brain barrier and increases brain microhemorrhages in mice and microbleeds in humans. Elevated urea alters the actin cytoskeleton and tight junction proteins in cultured endothelial cells, suggesting that these mechanisms explain (at least in part) the microhemorrhages and microbleeds observed in the animal and human studies.

A method combining SPE and Ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS-MS) was developed for the simultaneous determination of nine polypeptide antibiotics in animal tissues. The samples were extracted by methanol and 0.1 mol/L hydrochloric acid (7:3, v/v), then cleaned with Alumina-A and n-hexane, and finally purified by hydrophilic-lipophilic balance (hydrophilic-lipophilic balance, HLB) cartridge. The separations were performed on a C8 column (100A, 150 mm×2 mm, 3 µm) using a mobile phase gradient with 0.2% formic acid and acetonitrile. Electrospray positive ionization and multi-reaction monitoring (MRM) mode were used for detection. The calibration curves for vancomycin, norvancomycin, enramycin A, enramycin B, and teicoplanin, (concentration range 2~200 μg/L), colistin A, colistin B and bacitracin A (concentration range 5~500 μg/L), virginiamycin M1 (concentration range 0.1~20 μg/L) were linear with correlation coefficients more than 0.99. The limits of quantification (LOQ, S/N=10) of 9 kinds of polypeptide antibiotics were varied from 1 µg/kg to 20 µg/kg, the detection limit of the method was 0.3 µg/kg to 6 µg/kg. The recovery experiments were performed with chicken, pork, liver and kidney samples spiked in the range of 1~200 μg/kg. The average recoveries ranged from 74.2% and 96.3% with the relative standard deviations (RSDs) of 4.3%~14.6% (n=6). The method is simple and rapid with high sensitivity and good reproducibility. Furthermore, this method is effective for the safety monitoring of polypeptide antibiotic residues in animal tissues.

Oral anticoagulants are a critical component of stroke prevention, but carry a risk of brain hemorrhage. These hemorrhagic complications tend to occur in elderly individuals, especially those with predisposing conditions such as cerebral amyloid angiopathy (CAA). Clinical evidence suggests that non-vitamin K antagonist oral anticoagulants are safer than traditional oral anticoagulants. We analyzed whether the anticoagulant dabigatran produces cerebral microhemorrhage (the pathological substrate of MRI-demonstrable cerebral microbleeds) or intracerebral hemorrhage in aged mice with and without hemorrhage-predisposing angiopathy. We studied aged (22 months old) Tg2576 (a model of CAA) and wild-type (WT) littermate mice. Mice received either dabigatran etexilate (DE) (Tg N = 7; WT N = 10) or vehicle (Tg N = 9; WT N = 7) by gavage for 4 weeks. Anticoagulation effects of DE were confirmed using thrombin time assay. No mice experienced intracerebral hemorrhage. Cerebral microhemorrhage analysis, performed using Prussian-blue and H&E staining, showed no significant change in either number or size of cerebral microhemorrhage in DE-treated animals. Analysis of biochemical parameters for endothelial activation (ICAM-1), blood-brain barrier disruption (IgG, claudin-5, fibrinogen), microglial activation (Iba-1), or astrocyte activation (GFAP) showed neither exacerbation nor protective effects of DE in either Tg2576 or WT mice. Our study provides histological and biochemical evidence that aged mice, with or without predisposing factors for brain hemorrhage, tolerate anticoagulation with dabigatran. The absence of dabigatran-induced intracerebral hemorrhage or increased frequency of acute microhemorrhage may provide some reassurance for its use in high-risk patient populations.

BACKGROUND:Cerebral microhemorrhages (CMH) are commonly found in the aging brain. CMH are also the neuropathological substrate of cerebral microbleeds (CMB), demonstrated on brain MRI. Recent studies demonstrate the importance of systemic inflammation in CMH development, but the relationships among inflammation, aging, and CMH development are not well-defined. In the current study, we hypothesized that the pathogenesis of inflammation-induced CMH in mice differs by age. METHODS:We studied young (3 months, n = 20) and old (18 months, n = 25) C57BL/6 mice injected with low-dose lipopolysaccharide (LPS; 1 mg/kg, i.p.) or saline at 0, 6, and 24 h. Seven days after the first LPS/saline injection, brains were harvested, sectioned, and stained with hematoxylin and eosin (H&E) and Prussian blue (PB) to estimate acute/fresh and sub-acute CMH development, respectively. The relationships between microglial/macrophage activation (ionized calcium-binding adapter molecule-1), astrocyte activation (glial fibrillary acidic protein), blood-brain barrier (BBB) disruption (brain immunoglobulin G), aging, and CMH development were examined using immunohistochemistry. RESULTS:Aging alone did not increase spontaneous H&E-positive CMH development but significantly increased the number, size, and total area of LPS-induced H&E-positive CMH in mice. LPS- and saline-treated aged mice had significantly larger PB-positive CMH compared with young mice, but the total area of PB-positive CMH was increased only in LPS-treated aged mice. Aged mice had significantly increased microglial/macrophage activation, which correlated with H&E- and PB-positive CMH development. Aged mice treated with LPS had significantly increased astrocyte activation and BBB disruption compared with young LPS-treated mice. CONCLUSIONS:Aging makes the brain more susceptible to inflammation-induced CMH in mice, and this increase in CMH with aging is associated with microglial/macrophage activation.

Proteomics in animal science as well as in other biological sciences is a significant tool in the post-genomic era. In proteomic studies the presence and relative abundance of expressed proteins of a cell, tissue or biological fluid is studied. Recently, the whole genome of more and more domestic animal species is known, but genes and the transcribed mRNA have no direct effect on biological systems as they are regulated by proteins, which explain the importance of proteomics. The most common tool in proteomic approach is the two-dimensional polyacrylamide gel electrophoresis (2D PAGE), when proteins are separated by their isoelectric point followed by their mass separation as a second dimension. In this study authors used different sample preparation and protein staining methods on meat, liver and blood plasma and carried out 2D PAGE experiments. The most appropriate sample preparation methods are described in this paper. We concluded that depletion of major proteins in plasma is required but not necessary for meat and liver samples.

BackgroundCerebral microbleeds (CMB) are MRI-demonstrable cerebral microhemorrhages (CMH) which commonly coexist with ischemic stroke. This creates a challenging therapeutic milieu, and a strategy that simultaneously protects the vessel wall and provides anti-thrombotic activity is an attractive potential approach. Phosphodiesterase 3A (PDE3A) inhibition is known to provide cerebral vessel wall protection combined with anti-thrombotic effects. As an initial step in the development of a therapy that simultaneously treats CMB and ischemic stroke, we hypothesized that inhibition of the PDE3A pathway is protective against CMH development.MethodsThe effect of PDE3A pathway inhibition was studied in the inflammation-induced and cerebral amyloid angiopathy (CAA)-associated mouse models of CMH. The PDE3A pathway was modulated using two approaches: genetic deletion of PDE3A and pharmacological inhibition of PDE3A by cilostazol. The effects of PDE3A pathway modulation on H&E- and Prussian blue (PB)-positive CMH development, BBB function (IgG, claudin-5, and fibrinogen), and neuroinflammation (ICAM-1, Iba-1, and GFAP) were investigated.ResultsRobust development of CMH in the inflammation-induced and CAA-associated spontaneous mouse models was observed. Inflammation-induced CMH were associated with markers of BBB dysfunction and inflammation, and CAA-associated spontaneous CMH were associated primarily with markers of neuroinflammation. Genetic deletion of the PDE3A gene did not alter BBB function, microglial activation, or CMH development, but significantly reduced endothelial and astrocyte activation in the inflammation-induced CMH mouse model. In the CAA-associated CMH mouse model, PDE3A modulation via pharmacological inhibition by cilostazol did not alter BBB function, neuroinflammation, or CMH development.ConclusionsModulation of the PDE3A pathway, either by genetic deletion or pharmacological inhibition, does not alter CMH development in an inflammation-induced or in a CAA-associated mouse model of CMH. The role of microglial activation and BBB injury in CMH development warrants further investigation.

Summary: Selenium (Se) is essential for human health, however, data on population Se status and agriculture‐nutrition‐health linkages are limited in sub‐Saharan Africa (SSA). The scoping review aims to identify linkages between Se in soils/crops, dietary Se intakes, and livestock and human Se status in SSA. Online databases, organisational websites and grey literature were used to identify articles. Articles were screened at title, abstract and full text levels using eligibility criteria. The search yielded 166 articles from which 112 were excluded during abstract screening and 54 full text articles were assessed for eligibility. The scoping review included 34 primary studies published between 1984 and 2021. The studies covered Se concentrations in soils (n = 7), crops (n = 9), animal tissues (n = 2), livestock (n = 3), and human Se status (n = 15). The evidence showed that soil/crop Se concentrations affected Se concentration in dietary sources, dietary Se intake and biomarkers of Se status. Soil types are a primary driver of human Se status and crop Se concentration correlates positively with biomarkers of Se dietary status. Although data sets of Se concentrations exist across the food system in SSA, there is limited evidence on linkages across the agriculture‐nutrition nexus. Extensive research on Se linkages across the food chain is warranted. [ABSTRACT FROM AUTHOR]

Sodium pentachlorophenate (PCP) is widely used as a herbicide, fungicide, or molluscicide. It is highly toxic, easily soluble in water, making it highly prone to diffusion and causing water and soil pollution. Through the food chain, it enters animal bodies and remains in food, causing toxicity to humans and animals. Therefore, establishing a rapid and simple detection method for PCP is crucial for human health and environmental protection. Herein, lanthanide metal Eu 3+ was introduced into UiO-66-(COOH) 2 by post-synthesis modification, and the nanomaterials prepared based on this method have the advantages of both UiO-66-(COOH) 2 and Eu 3+ . The rigid skeleton structure of UiO-66-(COOH) 2 can protect the activity of antibody, the detection environment pH tolerance range of UiO-66-Eu is 3-11. While Eu 3+ has long fluorescence lifetime, high fluorescence intensity, high signal-to-noise ratio, and low photobleaching rate. UiO-66-Eu-based immunochromatography assay was successfully applied in PCP detection with the detection limits of 0.84, 0.98, and 0.37 μg/kg for pork, chicken, and shrimp, respectively, which was up to 10-fold more sensitive than the reported ICAs. The recoveries ranged from 79.7 %-113.1 %, with the coefficient of variation from 6.6 %-17.1 %. Parallel detection of 30 samples by LC-MS/MS showed a good correlation with that of our proposed method (R 2 >0.98). This work not only provides a creative attempt for UiO-66-Eu based highly sensitive and strongly tolerant ICAs, but also guarantees human health and environmental protection., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The exploitation of silver nanoparticles (AgNPs) in biomedicine represents more than one third of their overall application. Despite their wide use and significant amount of scientific data on their effects on biological systems, detailed insight into their in vivo fate is still lacking. This study aimed to elucidate the biotransformation patterns of AgNPs following oral administration. Colloidal stability, biochemical transformation, dissolution, and degradation behaviour of different types of AgNPs were evaluated in systems modelled to represent biological environments relevant for oral administration, as well as in cell culture media and tissue compartments obtained from animal models. A multimethod approach was employed by implementing light scattering (dynamic and electrophoretic) techniques, spectroscopy (UV–vis, atomic absorption, nuclear magnetic resonance) and transmission electron microscopy. The obtained results demonstrated that AgNPs may transform very quickly during their journey through different biological conditions. They are able to degrade to an ionic form and again reconstruct to a nanoparticulate form, depending on the biological environment determined by specific body compartments. As suggested for other inorganic nanoparticles by other research groups, AgNPs fail to preserve their specific integrity in in vivo settings.

BackgroundCerebral microhemorrhages (CMH) are tiny deposits of blood degradation products in the brain and are pathological substrates of cerebral microbleeds. The existing CMH animal models are β-amyloid-, hypoxic brain injury-, or hypertension-induced. Recent evidence shows that CMH develop independently of hypoxic brain injury, hypertension, or amyloid deposition and CMH are associated with normal aging, sepsis, and neurodegenerative conditions. One common factor among the above pathologies is inflammation, and recent clinical studies show a link between systemic inflammation and CMH. Hence, we hypothesize that inflammation induces CMH development and thus, lipopolysaccharide (LPS)-induced CMH may be an appropriate model to study cerebral microbleeds.MethodsAdult C57BL/6 mice were injected with LPS (3 or 1 mg/kg, i.p.) or saline at 0, 6, and 24 h. At 2 or 7 days after the first injection, brains were harvested. Hematoxylin and eosin (H&E) and Prussian blue (PB) were used to stain fresh (acute) hemorrhages and hemosiderin (sub-acute) hemorrhages, respectively. Brain tissue ICAM-1, IgG, Iba1, and GFAP immunohistochemistry were used to examine endothelium activation, blood-brain barrier (BBB) disruption, and neuroinflammation. MRI and fluorescence microscopy were used to further confirm CMH development in this model.ResultsLPS-treated mice developed H&E-positive (at 2 days) and PB-positive (at 7 days) CMH. No surface and negligible H&E-positive CMH were observed in saline-treated mice (n = 12). LPS (3 mg/kg; n = 10) produced significantly higher number, size, and area of H&E-positive CMH at 2 days. LPS (1 mg/kg; n = 9) produced robust development of PB-positive CMH at 7 days, with significantly higher number and area compared with saline (n = 9)-treated mice. CMH showed the highest distribution in the cerebellum followed by the sub-cortex and cortex. LPS-induced CMH were predominantly adjacent to cerebral capillaries, and CMH load was associated with indices of brain endothelium activation, BBB disruption, and neuroinflammation. Fluorescence microscopy confirmed the extravasation of red blood cells into the brain parenchyma, and MRI demonstrated the presence of cerebral microbleeds.ConclusionsLPS produced rapid and robust development of H&E-positive (at 2 days) and PB-positive (at 7 days) CMH. The ease of development of both H&E- and PB-positive CMH makes the LPS-induced mouse model suitable to study inflammation-induced CMH.

Highly mineralized collagen-containing beef raw materials (tibia, fibula and costal bones) are a source of valuable protein nutraceuticals. They include high molecular weight proteins, oligopeptides and amino acids, which anabolic and physiological potential is used insufficiently. Protein nutraceuticals were obtained by high-temperature hydrolysis of beef raw materials in combination with enzymolysis by proteolytic enzyme preparations Alcalase 2,5 L, Protamex, Protosubtilin G3x. The water-soluble fraction of hydrolysates was studied after its separation and freeze-drying on the content of nitrogenous compounds, fats, minerals, formol-titrated nitrogen, fractional molecular composition. The mathematical dependencies of accumulation of low molecular weight products of protein hydrolysis on enzymolysis duration and doses of different enzyme preparations were obtained. The rational technological scheme of complex processing of beef raw materials with production of protein, fat and mineral-protein additives was proposed. The protein weight fraction in the freeze-dried protein hydrolysates was 69.5–89.6%. All studied protein additives contained peptides with a molecular weight of not more than 100 kDa. The content of low-molecular weight oligopeptides with a molecular weight of less than 10 kDa in the protein additives obtained by enzymatic thermal hydrolysis was more than 90%. The amino acid composition of protein additives produced by different hydrolytic methods was analyzed. Sensory and functional-technological properties of freeze-dried protein compositions were studied. The amino acid potential, high assimilability and physiological activity of protein nutraceuticals from collagen-containing beef raw materials were established. It is recommended to use them in the composition of specialized biologically active additives (BAAs) to food of the osteotropic direction in recipes of specialized and personalized products as a source of amino acids and active peptides.

• Description of a new representation of non-targeted LC-MS data: Molecular Barcode™. • Similarity analysis of soy-, dairy- and animal tissue-based food products. • Rationale of a semi-targeted method to detect animal tissue. • Detection of animal tissue in food products, using p G+ and related fragments. • Discussion about possible applications of the presented convergent approach. There are various analytical techniques available to address the growing interest in the composition of food products. LC-HRMS(/MS) is the most comprehensive technique, providing detailed information at the molecular level. However, given the vast number of different molecules encountered in food products, it is important to obtain a global overview of the dataset before focusing on similarities and differences. Therefore, a convergent strategy was employed, going from non-targeted to targeted analysis, with insightful data representations, most notably Molecular Barcode. Additionally an intermediate, semi-targeted analysis was defined, aimed at the specific detection of animal tissue in food products, using p G+ and related fragments after all ion fragmentation. The use of Molecular Barcode as a starting point to obtain relevant molecular data was also demonstrated. In conclusion, the convergent approach facilitates the design of suitable targeted methods, either to discriminate between samples or to find a generic target. [ABSTRACT FROM AUTHOR]

One of the most widely used solutions to fix and preserve organic tissues is formaldehyde, despite reservations regarding its toxicity and the fact that formaldehyde‐embalmed bodies lose their original characteristics. Anatomy laboratories have been replacing formaldehyde with solutions that retain the characteristics of fresh tissue. For this purpose, alternative solutions with a very low concentration of formaldehyde or without any formaldehyde have been analyzed. The objective of this study was to compare biometry, coloration, haptic properties, and bacterial load on animal specimens (pig kidneys) embalmed with formaldehyde, and with Chilean Conservative Fixative Solution with and without formaldehyde (formaldehyde chCFS and formaldehyde‐free chCFS). Also, the perception of health and biological science students toward specimens treated with different solutions was assessed. The results indicated that there were no significant differences in specimens' retraction, or bacterial load. Students showed a preference for organs embalmed in formaldehyde chCFS and formaldehyde‐free chCFS; indicating that with these treatments they could better visualize structures and that the prosections had greater flexibility and the colors were more similar to those of fresh tissue. Additionally, students recommended the material embalmed in formaldehyde chCFS and formaldehyde‐free chCFS for anatomy learning. In contrast, students indicated that formaldehyde‐fixation negatively affected their practical experience. In conclusion, embalming with formaldehyde chCFS or formaldehyde‐free chCFS provides an advantageous practical experience over the use of formaldehyde and may be an alternative to replace the use of formaldehyde in anatomy laboratories. [ABSTRACT FROM AUTHOR]

Objective: The purpose of this work is to study whether the active state and species of biological tissues can influence changes in their dielectric properties. Methods: In this paper, the dielectric properties of liver, kidney and spleen tissues from human active, human inactive and animal tissues are measured in the frequency range of 10 Hz to 100 MHz. The four- and two-electrode methods are used to measure dielectric properties at different frequencies. Statistical analysis and the pattern recognition method are used to compare the dielectric properties of human active tissues, human inactive tissues, animal tissues and data provided by the IFAC database. Results: The results show that the dielectric properties of human active tissues are significantly different from those of human inactive tissues and animal tissues, resulting in a great difference between the dielectric properties provided by the IFAC database and those of human active tissues. The dielectric properties of human active tissues can be identified by the pattern recognition method based on principal component analysis, which further proves that the dielectric properties of human active tissues cannot be replaced. Conclusion: The dielectric properties of biological tissues are closely related to the activity and species of tissues. The dielectric properties of human active tissues cannot be replaced by those of human cadaver tissues or animal tissues. Significance: The significance of this study is suggesting that the IFAC database should be updated with the dielectric properties of human active tissues to provide accurate data for bioelectromagnetics research. [ABSTRACT FROM AUTHOR]

Cerebral microbleeds are microscopic hemorrhages with deposits of blood products in the brain, which can be visualized with MRI and are implicated in cerebrovascular diseases. Hematoxylin and eosin (H&E) and Perl's Prussian blue are popular staining methods used to localize cerebral microbleeds in pathology. This paper compared these two staining techniques in a mouse model of cerebral microbleeds. We used lipopolysaccharide (LPS) to induce cerebral microhemorrhages. C57B6 mice were treated with LPS (5 mg/kg, i.p.) or vehicle at baseline and at 24 hr. The brains were extracted 48 hr after the first injection and adjacent coronal sections were stained with H&E and Prussian blue to compare the effectiveness of the two staining techniques. H&E-positive stains were increased with LPS treatment and were correlated with grossly visible microhemorrhages on the brain surface; Prussian blue-positive stains, by comparison, showed no significant increase with LPS treatment and did not correlate with either H&E-positive stains or surface microhemorrhages. H&E staining is thus a more reliable indicator of acute bleeding events induced by LPS in this model within a short time span.

The application of histological methods in meat composition analysis and identification of prohibited tissues and organs added to meat and meat products is still in the research phase, although there have been some promising results. The aim of this work was to assess the possibility of using histological and histochemical methods for analysis of kulen composition. In this research, six samples of kulen were examined, one of which was produced in domestic conditions, while the rest were commercial products sampled from local markets. The samples were carried through classical histological preparation. The obtained slides were stained with haematoxylin/eosin, Masson-Goldner, toluidine blue and periodic acid-Schiff/alcian blue. The content of muscle, fat and connective tissue was evaluated using histomorphometric analysis. Histological analysis of kulen composition determined the presence of the following structures: muscle, adipose and connective tissues, blood vessels, glandular epithelium, peripheral nerve, cartilage and plant tissue. The histomorphometric analysis showed that the kulen products contained on average 56±2.52% muscle tissue, 7.27±1.38% connective tissue and 19.82±3.24% adipose tissue. The results show that by applying histological methods it is possible to identify different permitted and prohibited animal tissues in kulen, and hence, it is possible to analyse the composition of kulen. It is also possible to confirm the presence of various plant tissues, but for their precise identification, additional histological methods are needed. [ABSTRACT FROM AUTHOR]

Endometriosis is characterized by the presence of endometrial glands and stroma in extrauterine sites. Our objective was to determine whether endometriotic lesions (ELs) from women with endometriosis have altered retinoid levels compared with their eutopic endometrium, and to test the hypothesis that defects in all-trans retinoic acid (ATRA) biosynthesis in EL is related to reduced expression of cellular retinol-binding protein type 1 (RBP1). Retinoids were evaluated by liquid chromatography-tandem mass spectrometry and high-performance liquid chromatography in eutopic endometrial biopsies (EBs) and ELs from 42 patients with pathologically confirmed endometriosis. The ATRA levels were reduced, whereas the retinol and retinyl ester concentrations were elevated in EL compared with EB tissue. Similar results were found in a mouse model of endometriosis that used green fluorescent protein-positive endometrial tissue injected into the peritoneum of syngeneic hosts to mimic retrograde menses. The ATRA biosynthesis in vitro in retinol-treated primary human endometrial stromal cell (ESC) cultures derived from ELs was reduced compared with that of ESCs derived from patient-matched EBs. Correspondingly, RBP1 expression was reduced in tissue and ESCs derived from EL versus EB. Rbp1(-/-) mice showed reduced endometrial ATRA concentrations compared with wild type, associated with loss of tissue organization and hypercellularity. These findings provide the first quantitative measurements of ATRA in human endometrium and endometriosis, demonstrating reduced ATRA in ectopic tissue and corresponding ESC cultures. Quantitation of retinoids in murine endometriosis and in Rbp1(-/-) mice supports the contention that impaired ATRA synthesis caused by reduced RBP1 promotes an "endometriosis phenotype" that enables cells to implant and grow at ectopic sites.

PurposeThe first genomewide association study indicated that variations in the phosphodiesterase 4D (PDE4D) gene confer risk for ischemic stroke. However, inconsistencies among the studies designed to replicate the findings indicated the need for further investigation to elucidate the role of the PDE4 pathway in stroke pathogenesis. Hence, we studied the effect of global inhibition of the PDE4 pathway in two rat experimental stroke models, using the PDE4 inhibitor rolipram. Further, the specific role of the PDE4D isoform in ischemic stroke pathogenesis was studied using PDE4D knockout rats in experimental stroke.MethodsRats were subjected to either the ligation or embolic stroke model and treated with rolipram (3mg/kg; i.p.) prior to the ischemic insult. Similarly, the PDE4D knockout rats were subjected to experimental stroke using the embolic model.ResultsGlobal inhibition of the PDE4 pathway using rolipram produced infarcts that were 225% (p

To monitor the levels of furaltadone in animal tissues, an immunochromatographic strip (immunostrip) based on quantum dot was developed for rapid detection of 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ), the metabolite of furaltadone. Four monoclonal antibodies (mAbs) against nitrophenyl derivative of AMOZ (2-NP-AMOZ) were generated from the hybridoma cell lines. Among them, the mAb 5C10/3A8 showed the highest sensitivity to 2-NP-AMOZ. Using an immunostrip reader, with the 2-NP-AMOZ as the target, the immunostrip had a liner range from 1 to 30 ng/mL, with 50% inhibition concentration of 5.22 ng/mL and limit of detection of 0.07 ng/mL. The recoveries for AMOZ ranged from 78.6% to 108.9%, with coefficients of variation below 12%. A good correlation (R2 = 0.9901) between the immunostrip and LC–MS/MS results in the tissues tested demonstrated the reliability of the immunostrip. These results indicate that proposed immunostrip is suitable for the rapid and quantitative detection of AMOZ residues in edible animal tissues.

In this study, it was aimed to determine the presence of animal tissues and plant materials in traditionally produced fermented sausages by using histological and immunohistochemical methods, and to determine the animal tissues that are not legally permitted. In the study, fermented sausage samples (n=18) produced by a traditional method in Mardin (Turkey) and sold in butcher shops between January and March in 2019 were used as a test material. Samples were taken from 5 different locations of each sausage for histological examinations, and routine histological tissue follow-up was performed. Paraffinized sections were stained with Hematoxylin eosin, Crossmon's trichrome and Immunohistochemical techniques. Striated muscle, collagen fibers, connective, adipose, nerve, glandular, cartilage, bone, lung, heart, spleen tissues, blood vessels, glandular epithelium and various plant materials were detected in the examined sections. Animal tissues that are not permitted but included in fermented sausages were determined in all samples. It was concluded that the identification of these tissues in fermented sausage contents individually is important and to examine fermented sausages by histological methods in terms of preventing fraud in addition to the analyses performed for the evaluation of microbiological and chemical quality parameters, and that foods produced locally and presented to consumption without being subjected for inspection may pose a health risk. [ABSTRACT FROM AUTHOR]

To monitor the levels of furaltadone in animal tissues, an immunochromatographic strip (immunostrip) based on quantum dot was developed for rapid detection of 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ), the metabolite of furaltadone. Four monoclonal antibodies (mAbs) against nitrophenyl derivative of AMOZ (2-NP-AMOZ) were generated from the hybridoma cell lines. Among them, the mAb 5C10/3A8 showed the highest sensitivity to 2-NP-AMOZ. Using an immunostrip reader, with the 2-NP-AMOZ as the target, the immunostrip had a liner range from 1 to 30 ng/mL, with 50% inhibition concentration of 5.22 ng/mL and limit of detection of 0.07 ng/mL. The recoveries for AMOZ ranged from 78.6% to 108.9%, with coefficients of variation below 12%. A good correlation (R2 = 0.9901) between the immunostrip and LC–MS/MS results in the tissues tested demonstrated the reliability of the immunostrip. These results indicate that proposed immunostrip is suitable for the rapid and quantitative detection of AMOZ residues in edible animal tissues. [ABSTRACT FROM AUTHOR]

Dissociated cultures from many species have been important tools for exploring factors that regulate structure and function of central neuronal synapses. We have previously shown that cells harvested from brains of late stage Drosophila pupae can regenerate their processes in vitro. Electrophysiological recordings demonstrate the formation of functional synaptic connections as early as 3 days in vitro (DIV), but no information about synapse structure is available. Here, we report that antibodies against pre-synaptic proteins Synapsin and Bruchpilot result in punctate staining of regenerating neurites. Puncta density increases as neuritic plexuses develop over the first 4 DIV. Electron microscopy reveals that closely apposed neurites can form chemical synapses with both pre- and postsynaptic specializations characteristic of many inter-neuronal synapses in the adult brain. Chemical synapses in culture are restricted to neuritic processes and some neurite pairs form reciprocal synapses. GABAergic synapses have a significantly higher percentage of clear core versus granular vesicles than non-GABA synapses. Gap junction profiles, some adjacent to chemical synapses, suggest that neurons in culture can form purely electrical as well as mixed synapses, as they do in the brain. However, unlike adult brain, gap junctions in culture form between neuronal somata as well as neurites, suggesting soma ensheathing glia, largely absent in culture, regulate gap junction location in vivo. Thus pupal brain cultures, which support formation of interneuronal synapses with structural features similar to synapses in adult brain, are a useful model system for identifying intrinsic and extrinsic regulators of central synapse structure as well as function.

Sample pretreatment is directly related to the accuracy and reproducibility of test results, simple and controllable sample pretreatment can greatly reduce analysis errors. Herein, MIL-88B–NH 2 was synthesized with a regular octahedral rigid framework structure and used as antibody carrier to develop an immunochromatographic assay (ICA) for the detection of sodium pentachlorophenol (PCP) in animal tissues. MIL-88B–NH 2 has superior molar absorption coefficient, ultrahigh antibody affinity, and extreme detection environmental tolerance, which were 1.57, 2.5, and 2 times higher than those of traditional colloidal gold, respectively. Therefore, even complex animal tissues can be detected by simple extraction without nitrogen blowing. Ultimately, the cut-off values of this method for pork, chicken and shrimp were 8/5/3 μg/kg, the limits of detection were 1.15/1.28/0.25 μg/kg. The recoveries ranged from 95.5% to 103.0%, with the coefficient of variation from 1.87% to 9.69%. A parallel analysis of 30 samples was confirmed by LC-MS/MS; the results showed a good correlation (R2 > 0.95), indicating the authenticity and reliability of the MIL-88B–NH 2 -ICA. This work provides a detection strategy with enhanced sensitivity and robustness and simplified sample pretreatment, which has broad application prospects. [Display omitted] • MIL-88B–NH 2 was synthesized with extreme detection environmental tolerance. • MIL-88B–NH 2 -ICA was firstly developed with simplified sample preparation for PCP in animal tissues. • The sensitivity of the proposed method was 3–100 times higher than that of the reported methods. [ABSTRACT FROM AUTHOR]

The excessive accumulation of antibiotic residue in animal-derived foods pose a threat to our heath. Therefore, it is necessary to establish a rapid, simple, effective, safe and highly sensitive lincomycin (LIN) residues detection method by high-performance liquid chromatography equipped with ultraviolet. Here, due to the weak UV absorption of LIN, the p -Toluenesulfonyl isocyanate (PTSI, has a strong UV absorption) was binded to the hydroxyl group of LINs by using pre-column derivatization method, improving the UV absorption of LIN. This 9-min-run analytical method detects and quantifies LIN residues with acceptable validation performance parameters in terms of sensitivity, selectivity, linearity, the limit of quantification, accuracy, and precision.Our result showed that the limits of detection and quantification for LIN in samples were 25–40 μg/kg and 40–60 μg/kg, respectively, under the optimized conditions. Furthermore, the LIN recoveries ranged from 72.4 % to 96.7 %; the determination coefficients were 0.9912–0.9996, and the relative standard deviations were 5.6 %-12.7 %. Therefore, the HPLC-UVD method for detecting LIN residues in animal-derived food products has been established. [ABSTRACT FROM AUTHOR]

In Drosophila, nicotinic acetylcholine receptors (nAChRs) mediate fast excitatory synaptic transmission in mushroom body Kenyon cells, a neuronal population involved in generation of complex behaviors, including responses to drugs of abuse. To determine whether activation of nAChRs can induce cellular changes that contribute to functional plasticity in these neurons, we examined nicotine-evoked responses in cells cultured from brains of late stage OK107-GAL4 pupae. Kenyon cells can be identified by expression of green fluorescent protein (GFP+). Nicotine activates alpha-bungarotoxin-sensitive nAChRs, causing a rapid increase in intracellular calcium levels in over 95% of the Kenyon cells. The nicotine-evoked calcium increase has a voltage-gated calcium channel (VGCC) dependent component and a VGCC-independent component that involves calcium influx directly through nAChRs. Thapsigargin treatment reduces the nicotine response consistent with amplification by calcium release from intracellular stores. The response to nicotine is experience-dependent: a short conditioning pulse of nicotine causes a transient 50% reduction in the magnitude of the response to a test pulse of nicotine when the interpulse interval is 4 h. This cellular plasticity is dependent on activation of the VGCC-component of the nicotine response and on cAMP-signaling, but not on protein synthesis. These data demonstrate that activation of nAChRs induces a calcium-dependent plasticity in Kenyon cells that could contribute to adult behaviors involving information processing in the mushroom bodies including responses to nicotine.

Donor leukocyte infusion (DLI) can induce graft-versus-leukemia (GvL) reactions in patients with chronic myeloid leukemia (CML) relapsing after allogeneic bone marrow transplantation (BMT), but the mechanisms of the antileukemic effect of DLI are unknown, and the procedure is complicated by graft-versus-host disease (GvHD) and graft failure. Here, we adapted a murine retroviral BMT model of Philadelphia(+) leukemia by combining allogeneic bone marrow (BM) from C57Bl/6 (H-2(b)) mice with BCR-ABL-transduced Balb/c (H-2(d)) BM, inducing mixed chimerism and myeloproliferative disease in recipients resembling relapse of CML following allogeneic BMT. Infusions of allogeneic splenocytes eliminated BCR-ABL-induced CML-like disease in the majority of mixed chimeras, with significant GvL effects mediated by both CD4(+) and CD4(-) cells. BCR-ABL-induced acute B-lymphoblastic leukemia was also eradicated by DLI in major histocompatibility complex (MHC)-mismatched chimeras. Most DLI-treated mice converted to full allogeneic chimerism but succumbed frequently to GvHD or graft failure. When MHC-matched B10.D2 (H-2(d)) mice were the allogeneic donors, CML-like disease was more resistant to DLI. These results suggest that depletion of CD8(+) cells from DLI could impair GvL against CML, while increased MHC disparity between donor and recipient may improve the responsiveness of Philadelphia(+) B-lymphoblastic leukemia to DLI.

To monitor the illegal used of furaltadone, a highly sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and fluorescence-linked immunosorbent assay (FLISA) based on a monoclonal antibody (mAb) were developed for the detection of 3-amino-5-methylmorpholino-2-oxazolidinone (AMOZ), the major metabolite of furaltadone in animal tissues. The highly specific mAb, which was very sensitive to a nitrophenyl derivative of AMOZ (2-NP-AMOZ) with IC50 values of 0.11 and 0.09 ng/mL for ic-ELISA and FLISA, respectively, was selected for the development of immunoassays. For both the ic-ELISA and FLISA for AMOZ-spiked experiments, acceptable recovery rates of 81.1–105.3% and coefficients of variation of 4.7–9.8% were obtained. In addition, results from both ic-ELISA and FLISA methods for spiked samples’ data showed excellent correlation coefficients ranging from 0.9652 to 0.9927. Meanwhile, the proposed ic-ELISA and FLISA for thirty spiked samples were confirmed by standard LC-MS/MS with high correlation coefficients of 0.9911 and 0.9921, respectively. These results suggest that the developed ic-ELISA and FLISA are valid and cost-effective tools for high-throughput monitoring methods for AMOZ residues in animal tissues.

Agrin mediates motor neuron-induced differentiation of the postsynaptic apparatus of the neuromuscular junction but its function in brain remains unknown. Here we report that expression of c-fos, induced by activation of nicotinic or glutamatergic receptors, was significantly lower in cortical neurons cultured from agrin-deficient mutant mouse embryos compared to wildtype. Agrin-deficient neurons also exhibited increased resistance to excitotoxic injury. Treatment with recombinant agrin restored glutamate-induced c-fos expression and excitotoxicity of the agrin-deficient neurons to near wild-type levels, confirming the agrin dependence of the phenotype. The observation that c-fos induction by activation of voltage-gated Ca2+ channels is also reduced in agrin-deficient neurons raises the possibility that agrin may play a wider role by regulating responses to Ca(2+)-mediated signals. Consistent with the decline in response of cultured mutant neurons to glutamate, decreases in kainic acid-induced seizure and mortality were observed in adult agrin heterozygous mice. Together, these data demonstrate that agrin plays an important role in defining neuronal responses to excitatory neurotransmitters both in vitro and in vivo.

Tyrosine kinase fusion oncogenes that occur as a result of chromosomal translocations have been shown to activate proliferative and antiapoptotic pathways in leukemic cells, but the importance of autocrine and paracrine expression of hematopoietic cytokines in leukemia pathogenesis is not understood. Evidence that leukemic transformation may be, at least in part, cytokine dependent includes data from primary human leukemia cells, cell culture experiments, and murine models of leukemia. This report demonstrates that interleukin (IL)-3 plasma levels are elevated in myeloproliferative disease (MPD) caused by the TEL/tyrosine kinase fusions TEL/platelet-derived growth factor beta receptor (PDGFbetaR), TEL/Janus kinase 2 (JAK2), and TEL/neurotrophin-3 receptor (TRKC). Plasma granulocyte-macrophage colony-stimulating factor (GM-CSF) levels were elevated by TEL/PDGFbetaR and TEL/JAK2. However, all of the fusions tested efficiently induced MPD in mice genetically deficient for both GM-CSF and IL-3, demonstrating that these cytokines are not necessary for the development of disease in this model system. Furthermore, in experiments using normal marrow transduced with TEL/PDGFbetaR retrovirus mixed with marrow transduced with an enhanced green fluorescent protein (EGFP) retrovirus, the MPD induced in these mice demonstrated minimal stimulation of normal myelopoiesis by the TEL/PDGFbetaR-expressing cells. In contrast, recipients of mixed GM-CSF-transduced and EGFP-transduced marrow exhibited significant paracrine expansion of EGFP-expressing cells. Collectively, these data demonstrate that, although cytokine levels are elevated in murine bone marrow transplant models of leukemia using tyrosine kinase fusion oncogenes, GM-CSF and IL-3 are not required for myeloproliferation by any of the oncogenes tested.

Reliable wireless communication inside the human body is crucial for the design of implantable body sensor networks. The tissues in human body are heterogeneous and have different conductivity and permittivity, which make the modeling of the wireless channel challenging. The design of upper layers of the network stack requires the physical layer characteristics including the channel model. Currently, there is no unique channel model available for implant communication inside body. Various measurement campaigns of channel characteristics are underway. The channel model characteristics depends on the hardware components used such as antenna and matching circuit as well as the operating frequency, which are not taken into account by the existing channel models for implant communication. Moreover, hardware losses and different tissue characteristics have not been taken into account in the link budget of the existing channel models. The approach used in this paper pays special attention to the losses introduced by hardware components of the implant itself and the physical medium. This paper presents characteristics of radio channel using animal tissue. A comparison is made between these measured characteristics and the existing channel characteristics provided by the IEEE 802.15.6 standard. The empirical measurements are used to validate the simulations of the IEEE 802.15.6 model.

The BCR/ABL oncogene results from a balanced translocation between chromosomes 9 and 22 and is found in patients with chronic myeloid leukemia (CML) and in some patients with acute B-lymphoid leukemia. The Bcr/Abl fusion protein is a constitutively active tyrosine kinase that stimulates several intracellular signaling pathways, including activation of Ras through direct binding of the SH2-containing adapter protein Grb2 to Bcr tyrosine 177. A tyrosine-to-phenylalanine mutation (Y177F) at this site blocks the co-association of Bcr/Abl and Grb2 in vivo and impairs focus formation by Bcr/Abl in fibroblasts. However, the Bcr/Abl Y177F mutant can transform hematopoietic cell lines and primary bone marrow cells in vitro, so the importance of the Bcr/Abl-Grb2 interaction to myeloid and lymphoid leukemogenesis in vivo is unclear. We have recently demonstrated the efficient induction of CML-like myeloproliferative disease by BCR/ABL in a murine bone marrow transduction/transplantation model system. The Y177F mutation greatly attenuates the myeloproliferative disease induced by BCR/ABL, with mice developing B- and T-lymphoid leukemias of longer latency. In addition, the v-abl oncogene of Abelson murine leukemia virus, whose protein product lacks interaction with Grb2, is completely defective for the induction of CML-like disease. These results suggest that direct binding of Grb2 is required for the efficient induction of CML-like myeloproliferative disease by oncogenic Abl proteins. (Blood. 2000;96:664-670)

Highlights • HILIC-MS/MS was used for the determination of 16 coccidiostats in muscle and eggs. • Good accuracy and remarkably low LOQs were achieved in very short analysis time. • 82 meat & egg samples were analysed through the Greek National Residue Control Plan. • New data were obtained regarding the occurrence of coccidiostat residues in Greece. Abstract A simple, sensitive and efficient confirmatory method was developed and validated for the determination of 16 coccidiostats in animal tissues and eggs using hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC-MS/MS). The sample preparation consisted of a solid-liquid extraction with ACN and dispersive SPE cleanup with MgSO 4 and C 18. Analysis was realized in an Acquity BEH HILIC silica column, in SRM mode. Both positive and negative ionization was performed, using polarity switching. Isocratic elution was used with a mobile phase of ACN: aqueous ammonium formate 1 mM with 0.1% formic acid (80:20, v/v). Method validation was performed in eggs, poultry, bovine, ovine, porcine and rabbit tissue and exceptionally low LODs were achieved, varying from 0.004 μg kg−1 (decoquinate in porcine tissue) to 0.560 μg kg−1 (halofuginone in eggs). The developed methodology was applied in 82 muscle and egg samples through the Greek National Residue Control Plan for coccidiostats. [ABSTRACT FROM AUTHOR]

Previous studies in postnatal mouse demonstrating high levels of alpha7 nicotinic acetylcholine receptors on layer IV somatosensory cortical neurons coincident with the onset of functional synaptic transmission led us to investigate whether the number and/or the localization of these receptors could be regulated by activity. Accordingly, we examined alpha-bungarotoxin binding in mouse somatosensory cortex following removal of all of the vibrissae on one side of the face, either by vibrissal follicle cauterization or daily plucking beginning on the day of birth. Following vibrissa plucking, the levels of [125I]alpha-bungarotoxin binding on postnatal day 6 were significantly higher (23 +/- 7%) in the denervated cortex (contralateral to the peripheral manipulation) than the intact cortex. Cauterization also resulted in significantly higher (14 +/- 3%) [125I]alpha-bungarotoxin binding in the contralateral vs. the ipsilateral cortex. In contrast, there was no difference in [125I]alpha-bungarotoxin binding in the left and right cortices of unoperated control animals. At postnatal day 14, levels of [125I]alpha-bungarotoxin binding in layer IV were very low in control animals as well as in animals subjected to whisker plucking or cautery. These findings suggest that reducing activity in the somatosensory pathway regulates the density of alpha7 nicotinic acetylcholine receptors during the first postnatal week. However, the normal decrease in receptor density that is seen during the second postnatal week of development proceeds despite altered sensory activity.

Agrin is an extracellular matrix protein involved in the formation of the postsynaptic apparatus of the neuromuscular junction. In addition to spinal motor neurons, agrin is expressed by many other neuronal populations throughout the nervous system. Agrin's role outside of the neuromuscular junction, however, is poorly understood. Here we use the polymerase chain reaction to examine expression and alternative splicing of agrin in mouse somatosensory cortex during early postnatal development in vivo and in dissociated cell culture. Peak levels of agrin gene expression in developing cortex coincide with ingrowth of thalamic afferent fibres and formation of thalamocortical and intracortical synapses. Analysis of alternatively spliced agrin messenger RNA variants shows that greater than 95% of all agrin in developing and adult somatosensory cortex originates in neurons, including isoforms that have little or no activity in acetylcholine receptor aggregation assays. The levels of expression of "active" and "inactive" isoforms, however, are regulated during development. A similar pattern of agrin gene expression is also observed during a period when new synapses are being formed between somatosensory neurons growing in dissociated cell culture. Changes in agrin gene expression, observed both in vivo and in vitro, are consistent with a role for agrin in synapse formation in the central nervous system.

Previous studies in rat, showing a transient pattern of expression of the alpha 7 nicotinic acetylcholine receptor in the ventrobasal thalamus and barrel cortex during the first 2 postnatal weeks, suggest that these receptors may play a role in development of the thalamocortical system. In the present study, in situ hybridization and radiolabeled ligand binding were employed to examine the spatiotemporal distribution of alpha 7 mRNA and alpha-bungarotoxin binding sites in the thalamocortical pathway of mouse during early postnatal development. As in the rat, high levels of alpha 7 mRNA and alpha-bungarotoxin binding sites are present in the barrel cortex of mouse during the first postnatal week. Both alpha 7 mRNA and its receptor protein are observed in all cortical laminae, with the highest levels seen in the compact cortical plate, layer IV, and layer VI. When viewed in a tangential plane, alpha 7 mRNA and alpha-bungarotoxin binding sites delineate a whisker-related barrel pattern in layer IV by P3-5. Quantitative analysis reveals a dramatic decrease in the levels of expression of alpha 7 mRNA and alpha-bungarotoxin binding sites in the cortex by the end of the second postnatal week. Unlike in the rat, only low levels of alpha 7 mRNA or alpha-bungarotoxin binding sites are present in the ventrobasal complex of the mouse thalamus. The broad similarities between the thalamocortical development of rat and mouse taken together with the present results suggest that alpha 7 receptors located on cortical neurons, rather than on thalamic neurons, play a role in mediating aspects of thalamocortical development.

TRH is synthesized from a larger 26-kilodalton (kDa) prohormone (pro-TRH). Rat pro-TRH contains five copies of the TRH progenitor sequence (Gln-His-Pro-Gly) and seven other cryptic peptides. Each of the five TRH progenitor sequences is flanked by pairs of basic amino acids. We used a bovine intermediate lobe secretory vesicle membrane preparation, which contains the prohormone convertases (PCs) PC1 and PC2, to study the in vitro processing of pro-TRH. Pro-TRH was radiolabeled using [3H]Leu in AtT20 cells transfected with prepro-TRH complementary DNA, and the labeled 26-kDa pro-TRH was isolated from the cell extract by preparative sodium dodecyl sulfate-gel electrophoresis. Incubation of [3H]pro-TRH with the intermediate lobe secretory vesicle membrane preparation was followed by immunoprecipitation with antibodies specific for various regions of the pro-TRH sequence, and the immunoprecipitates were analyzed by sodium dodecyl sulfate-gel electrophoresis. Immunoprecipitation of the reaction mixture with anti-pCC10 antibody (an antibody that recognizes the intact precursor and amino-terminal intermediate products of processing) showed a time-dependent appearance of a 15-kDa and a 6-kDa peptide and, at times, a 3.8-kDa peptide with diminution of the 26-kDa substrate. Immunoprecipitation of the incubate with the C-terminal-directed antibody, pYE17 (an antibody that recognizes the intact precursor and C-terminal intermediate products of processing), showed the generation of 16.5-, 10-, and 5.4-kDa products in a time-dependent manner, with disappearance of the substrate. Western blot analysis demonstrated that the secretory vesicle membrane preparation contains PC1 and PC2. Immunodepletion studies with antiserum specific for PC1 or PC2 demonstrated that PC1 and PC2 can process pro-TRH to these intermediate products. An initial site of cleavage appeared to be either at the 152-153 or the 158-159 pair of basic residues to yield a 15-kDa N-terminal fragment that was then processed to the 6-kDa [TRH-(25-74)] and 3.8-kDa [TRH-(83-112)] forms. The 10-kDa C-terminal peptide generated by this cleavage was then processed to a 5.4-kDa peptide [TRH-(208-255)]. Alternatively, an initial cleavage at the 107-108 or the 112-113 bonds was also observed, yielding a 16.5-kDa C-terminal product that was further processed to the 5.4-kDa peptide. The pH profile for the appearance of both C- and N-terminal products showed a bimodal distribution, with optima at both 5.5 and 7.5. The cleavage of pro-TRH was enhanced by Ca2+ and partially inhibited by Zn2+.(ABSTRACT TRUNCATED AT 400 WORDS)