The role of glial autophagy in Alzheimer's disease.

Although Alzheimer's disease is the most pervasive neurodegenerative disorder, the mechanism underlying its development is still not precisely understood. Available data indicate that pathophysiology of this disease may involve impaired autophagy in glial cells. The dysfunction is manifested as reduced ability of astrocytes and microglia to clear abnormal protein aggregates. Consequently, excessive accumulation of amyloid beta plaques and neurofibrillary tangles activates microglia and astrocytes leading to decreased number of mature myelinated oligodendrocytes and death of neurons. These pathologic effects of autophagy dysfunction can be rescued by pharmacological activation of autophagy. Therefore, a deeper understanding of the molecular mechanisms involved in autophagy dysfunction in glial cells in Alzheimer's disease may lead to the development of new therapeutic strategies. However, such strategies need to take into consideration differences in regulation of autophagy in different types of neuroglia.

Graphene oxide down-regulates genes of the oxidative phosphorylation complexes in a glioblastoma.

BACKGROUND: Recently different forms of nanographene were proposed as the material with high anticancer potential. However, the mechanism of the suppressive activity of the graphene on cancer development remains unclear. We examined the effect of oxygenated, reduced and pristine graphene on the gene expression in glioblastoma U87 cell line. RESULTS: Conducting microarrays and RT-qPCR analysis we explored that graphene oxide (rather than reduced graphene oxide and pristine graphene) down-regulates the mRNA expression of mitochondrial oxidative phosphorylation (OXPHOS) nuclear genes of complexes I, III, IV and V. The presented results provide first evidence for the hypothesis that the suppressed growth of GBM can be the consequence of down-regulation of OXPHOS protein expression and decreased ATP level. CONCLUSIONS: We suggest that changes in the expression of OXPHOS genes identified in our study may mediate the anti-proliferative and anti-migratory effects of graphene oxide in glioblastoma cells. However, further investigations with different cell lines, regarding expression, regulation and activity of OXPHOS genes identified in our study is necessary to elucidate the mechanism mediating the anti-proliferative and anti-migratory effects of graphene oxide in glioblastoma cells.

Unappreciated Role of LDHA and LDHB to Control Apoptosis and Autophagy in Tumor Cells.

Tumor cells possess a high metabolic plasticity, which drives them to switch on the anaerobic glycolysis and lactate production when challenged by hypoxia. Among the enzymes mediating this plasticity through bidirectional conversion of pyruvate and lactate, the lactate dehydrogenase A (LDHA) and lactate dehydrogenase B (LDHB), are indicated. LDHA has a higher affinity for pyruvate, preferentially converting pyruvate to lactate, and NADH to NAD+ in anaerobic conditions, whereas LDHB possess a higher affinity for lactate, preferentially converting lactate to pyruvate, and NAD+ to NADH, when oxygen is abundant. Apart from the undisputed role of LDHA and LDHB in tumor cell metabolism and adaptation to unfavorable environmental or cellular conditions, these enzymes participate in the regulation of cell death. This review presents the latest progress made in this area on the roles of LDHA and LDHB in apoptosis and autophagy of tumor cells. Several examples of how LDHA and LDHB impact on these processes, as well as possible molecular mechanisms, will be discussed in this article. The information included in this review points to the legitimacy of modulating LDHA and/or LDHB to target tumor cells in the context of human and veterinary medicine.

Glioblastoma multiforme - an overview.

Glioblastoma multiforme is a central nervous system tumor of grade IV histological malignancy according to the WHO classification. Over 90% of diagnosed glioblastomas multiforme cases are primary gliomas, arising from normal glial cells through multistep oncogenesis. The remaining 10% are secondary gliomas originating from tumors of lower grade. These tumors expand distinctly more slowly. Although genetic alterations and deregulations of molecular pathways leading to both primary and secondary glioblastomas formation differ, morphologically they do not reveal any significant differences. Glioblastoma is a neoplasm that occurs spontaneously, although familial gliomas have also been noted. Caucasians, especially those living in industrial areas, have a higher incidence of glioblastoma. Cases of glioblastoma in infants and children are also reported. The participation of sex hormones and viruses in its oncogenesis was also suggested. Progression of glioblastoma multiforme is associated with deregulation of checkpoint G1/S of a cell cycle and occurrence of multiple genetic abnormalities of tumor cells. Metastases of glioblastoma multiforme are rarely described. Treatment of glioblastoma multiforme includes tumor resection, as well as radiotherapy and chemotherapy. Drugs inhibiting integrin signaling pathways and immunotherapy are also employed. Treatment modalities and prognosis depend on the tumor localization, degree of its malignancy, genetic profile, proliferation activity, patient's age and the Karnofsky performance scale score. Although the biology of glioblastoma multiforme has recently been widely investigated, the studies summarizing the knowledge of its development and treatment are still not sufficient in terms of comprehensive brain tumor analysis.

Does ß-Hydroxy-ß-Methylbutyrate Have Any Potential to Support the Treatment of Duchenne Muscular Dystrophy in Humans and Animals?

Skeletal muscle is the protein reservoir of our body and an important regulator of glucose and lipid homeostasis. The dystrophin gene is the largest gene and has a key role in skeletal muscle construction and function. Mutations in the dystrophin gene cause Duchenne and Becker muscular dystrophy in humans, mice, dogs, and cats. Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular condition causing progressive muscle weakness and premature death. ß-hydroxy ß-methylbutyrate (HMB) prevents deleterious muscle responses under pathological conditions, including tumor and chronic steroid therapy-related muscle losses. The use of HMB as a dietary supplement allows for increasing lean weight gain; has a positive immunostimulatory effect; is associated with decreased mortality; and attenuates sarcopenia in elderly animals and individuals. This study aimed to identify some genes, metabolic pathways, and biological processes which are common for DMD and HMB based on existing literature and then discuss the consequences of that interaction.

Mycobacterial Interspersed Repeat Unit-Variable Number Tandem Repeat Typing of Mycobacterium avium Strains Isolated from the Lymph Nodes of Free-Living Carnivorous Animals in Poland.

Non-tuberculous mycobacteria (NTM) are ubiquitous organisms, of which some, especially those of the Mycobacterium avium complex (MAC), may be opportunistic animal and human pathogens. Infection with NTM can interfere with tuberculosis (TB) diagnosis and induce zoonoses, especially in immunocompromised individuals. Diseases caused by NTM have become more readily recognized; however, they are likely still underestimated. In this study, we identified and genotyped Mycobacterium avium strains that were isolated during TB monitoring among free-living carnivorous animals from southeastern Poland. In 2011-2020, lymph node samples from 192 such animals were tested for mycobacteria. A total of 41 isolates of M. avium strains were detected with the use of IS901, IS900, IS1245, and mycobacterial interspersed repeat unit-variable number tandem repeat (MIRU-VNTR) identification. Thirty-three were identified as M. avium subsp. avium. These strains were derived from 1 beech marten (Martes foina), 1 common buzzard (Buteo buteo), 2 European badgers (Meles meles), 3 wolves (Canis lupus), and 26 red foxes (Vulpes vulpes). One strain isolated from a wolf was identified as M. avium subsp. hominissuis. The results show the widespread occurrence of MAC bacilli in the studied environment and additionally comprise new data on the molecular characteristics of M. avium subspecies carried by free-living southeastern Polish carnivores.

A serological survey of Toxoplasma gondii in polish pigs from organic farms, other housing systems and in pigs of different age groups.

BACKGROUND: The consumption of raw or undercooked meat, especially pork, and offal containing infective tissue cysts is suspected to be a significant route of infection with Toxoplasma gondii. Although the use of "animal-friendly pig production systems" ensuring direct contact with the natural environment offers ethical benefits, it limits the ability to ensure animal health; it may also increase the probability of infections by pathogens such as T. gondii, and thus their entry into the food chain. This study determines the seroprevalence of T. gondii in pigs from different housing systems and farms with different hygiene standards in Poland, as well as among pigs of different age groups from farms with high hygiene standards. In total 760 pig serum samples were examined for the presence of specific antibodies using the PrioCHECK® Toxoplasma Ab porcine commercial ELISA test (Prionics, Switzerland). RESULTS: Test results with PP ≥ 20% were regarded as positive, as indicated by the manufacturer. Antibodies to T. gondii were found in 193 of 760 (25.4%) tested sera. Regarding different housing systems, antibodies were found in 117 pigs: of these, 52.6% (61/116) were from organic farms, 40.9% (47/115) from farms with low hygiene standards, 5.4% (9/167) from farms with high hygiene standards and 0% (0/40) from a farm with a high level of biosecurity. Regarding age groups, antibodies were found in 76 animals on farms with high hygiene standards: 11.1% (7/63) were pigs younger than 3 months, 0% (0/60) aged 3-4 months, 12.3% (7/57) aged 5-6 months (final fattening stage) and 43.7% (62/142) were sows aged 9 months and older. CONCLUSIONS: Antibodies to T. gondii were most often found in pigs from organic and low-hygiene farms, as well as in pigs aged 9 months and older. Meat derived from seropositive animals can pose a potential source of infection for humans. As maternal antibodies to T. gondii can be present in the blood of piglets aged up to 3-4 months, serological examination is unjustified in piglets up to this age.

The Secrets of Alternative Autophagy.

For many years, it was thought that ATG5 and ATG7 played a pivotal role in autophagy, and that the knockdown of one of these genes would result in its inhibition. However, cells with ATG5 or ATG7 depletion still generate autophagic vacuoles with mainly trans-Golgi-originated isolation membranes and do not die. This indicates that autophagy can occur via ATG5/ATG7-independent alternative autophagy. Its molecular mechanism differs from that of the canonical pathway, including inter alia the phosphorylation of ULK1, and lack of LC3 modifications. As the alternative autophagy pathway has only recently been described, little is known of its precise role; however, a considerable body of evidence suggests that alternative autophagy participates in mitochondrion removal. This review summarizes the latest progress made in research on alternative autophagy and describes its possible molecular mechanism, roles and methods of detection, and possible modulators. There is a need for further research focused on types of autophagy, as this can elucidate the functioning of various cell types and the pathogenesis of human and animal diseases.

New aspects of the esophageal histology of the domestic goat (Capra hircus) and European roe deer (Capreolus capreolus).

The present study examines the esophageal wall of animals from two distinct families of the Ruminantia: domestic goats and European roe deer. Five fragments were collected from the entire length of the esophageal wall in five goats and four roe deer and subjected to microscopic and morphometric analyses. All layers of the esophageal wall except the tela submucosa were found to be thicker in the goats. In both species, the esophagus was lined by parakeratinized stratified squamous epithelium, and the tela submucosa was deprived of glands along its entire length. However, the structure of the lamina muscularis mucosae was better developed in goats: it was found to be discontinuous in the proximal part, and then became fused in the cervical part, that is around the most proximal quarter of its length. In contrast, in roe deer, the lamina muscularis mucosae began as sparse, thin muscle bundles at the pharyngeal-esophageal junction, which thickened and clustered further down the esophagus, but did not fuse. Our findings regarding the microscopic structure of the ruminant esophagus are not fully consistent with the widely-accepted view and suggest that the histological structure of the esophagus demonstrates interspecies variation within this large suborder. More precisely, species-specific differences can be seen regarding the presence of esophageal glands and parakeratinized epithelium, and in the organization of the lamina muscularis mucosae.

[Regulation of apoptosis in endometrium preparation for menstruation or embryo implantation]. / Regulacja apoptozy w procesie przygotowania endometrium do menstruacji lub implantacji zarodka.

Recent studies have presented apoptosis as the key mechanism of endometrial tissue reconstruction. Regulation of apoptosis leads to the menstruation or prepare the mucose layer for the implantation of the embryo. Thus, the factors controlling apoptosis determine proper endometrial preparation for blastocyst implantation. Among these factors, not only the progesterone but also embryonic factors (chorion gonadotropin) and those produced by endometrium affected by interaction with blastocyst (prolactin, IGFBP-1) plays the pivotal role.

Geranylgeraniol Prevents Statin-Dependent Myotoxicity in C2C12 Muscle Cells through RAP1 GTPase Prenylation and Cytoprotective Autophagy.

The present study investigated the cytotoxic effects of statins (atorvastatin (ATR) and simvastatin (SIM), resp.) and methyl-beta-cyclodextrin (MßCD), at their respective IC50 concentrations, on muscle regeneration in the in vitro model of murine C2C12 myoblasts. Cotreatment with mevalonate (MEV), farnesol (FOH), geranylgeraniol (GGOH), or water-soluble cholesterol (Chol-PEG) was employed to determine whether the statin-dependent myotoxicity resulted from the lower cholesterol levels or the attenuated synthesis of intermediates of mevalonate pathway. Our findings demonstrated that while GGOH fully reverted the statin-mediated cell viability in proliferating myoblasts, Chol-PEG exclusively rescued MßCD-induced toxicity in myocytes. Statins caused loss of prenylated RAP1, whereas the GGOH-dependent positive effect was accompanied by loss of nonprenylated RAP1. Geranylgeranyltransferases are essential for muscle cell survival as inhibition with GGTI-286 could not be reversed by GGOH cotreatment. The increase in cell viability correlated with elevated AKT 1(S463) and GSK-3ß(S9) phosphorylations. Slight increase in the levels of autophagy markers (Beclin 1, MAP LC-3IIb) was found in response to GGOH cotreatment. Autophagy rose time-dependently during myogenesis and was inhibited by statins and MßCD. Statins and MßCD also suppressed myogenesis and neither nonsterol isoprenoids nor Chol-PEG could reverse this effect. These results point to GGOH as the principal target of statin-dependent myotoxicity, whereas plasma membrane cholesterol deposit is ultimately essential to restore viability of MßCD-treated myocytes. Overall, this study unveils for the first time a link found between the GGOH- and Chol-PEG-dependent reversal of statin- or MßCD-mediated myotoxicity and cytoprotective autophagy, respectively.

Immunohistochemical characteristic of C cells in European bison thyroid gland.

INTRODUCTION: C cells constitute a small percentage of thyroid gland parenchyma. The number, morphology and distribution of C cells differ among species; however, data regarding their characteristics in European bison are sparse. The aim of this study was to evaluate the morphology, distribution pattern and percentage of C cells in European bison thyroid gland together with morphometric analysis. MATERIAL AND METHODS: Thyroid glands from 28 European bisons of different sex and age were collected either in autumn-winter (13/28) or in spring-summer (15/28) periods and analyzed by immunohistochemistry. RESULTS: The mean total C cell number per all endocrine (follicular and C cells) cell number (C cell concentration) was 7.33%. The tendency to increase the C cell number from periphery to the central region of thyroid lobe was observed with the mean C cell concentration of 3.95%, 7.89% and 9.97% in peripheral, intermediate and central areas, respectively. Most frequently, C cells were situated intrafolliculary whereas epifollicular and interfollicular positions were observed less often. C cells were polymorphic with long cytoplasmic processes. The mean C cell area was 61.97 µm2 and the mean C cell perimeter, length and width were: 34.92 µm, 12.85 µm and 4.91 µm, respectively. In the majority of C cells, strong immunohistochemical cytoplasmic reaction was observed with the mean color intensity of 78.32. In autumn-winter period, C cells were significantly larger with lower color intensity than during spring and summer. CONCLUSIONS: This study leads to deeper characteristics of thyroid gland C cells in European bison. The histomorphometric data suggest that in European bison production of calcitonin by C cells may differ depending on the time of the year.

Verapamil treatment induces cytoprotective autophagy by modulating cellular metabolism.

Verapamil, an L-type calcium channel blocker, has been used successfully to treat cardiovascular diseases. Interestingly, we have recently shown that treatment of cancer cells with verapamil causes an effect on autophagy. As autophagy is known to modulate chemotherapy responses, this prompted us to explore the impact of verapamil on autophagy and cell viability in greater detail. We report here that verapamil causes an induction of autophagic flux in a number or tumor cells and immortalized normal cells. Moreover, we found that inhibition of autophagy in COLO 205 cells, via treatment with the chloroquine (CQ) or by CRISPR/Cas9-mediated disruption of the autophagy genes Atg7 and Atg5, causes an upregulation of apoptotic markers in response to verapamil. In search of a mechanism for this effect and because autophagy can often mitigate metabolic stress, we examined the impact of verapamil on cellular metabolism. This revealed that in normal prostate cells, verapamil diminishes glucose and glycolytic intermediate levels leading to adenosine 5'-triphosphate (ATP) depletion. In contrast, in COLO 205 cells it enhances aerobic glycolysis and maintains ATP. Importantly, we found that the autophagic response in these cells is related to the activity of l-lactate dehydrogenase A (LDHA, EC 1.1.1.27), as inhibition of LDHA reduces both basal and verapamil-induced autophagy and consequently decreases cell viability. In summary, these findings not only identify a novel mechanism of cytoprotective autophagy induction but they also highlight the potential of using verapamil together with inhibitors of autophagy for the treatment of malignant disease. ENZYMES: l-lactate dehydrogenase (LDHA, EC 1.1.1.27).

A homozygous FANCM mutation underlies a familial case of non-syndromic primary ovarian insufficiency.

Primary Ovarian Insufficiency (POI) affects ~1% of women under forty. Exome sequencing of two Finnish sisters with non-syndromic POI revealed a homozygous mutation in FANCM, leading to a truncated protein (p.Gln1701*). FANCM is a DNA-damage response gene whose heterozygous mutations predispose to breast cancer. Compared to the mother's cells, the patients' lymphocytes displayed higher levels of basal and mitomycin C (MMC)-induced chromosomal abnormalities. Their lymphoblasts were hypersensitive to MMC and MMC-induced monoubiquitination of FANCD2 was impaired. Genetic complementation of patient's cells with wild-type FANCM improved their resistance to MMC re-establishing FANCD2 monoubiquitination. FANCM was more strongly expressed in human fetal germ cells than in somatic cells. FANCM protein was preferentially expressed along the chromosomes in pachytene cells, which undergo meiotic recombination. This mutation may provoke meiotic defects leading to a depleted follicular stock, as in Fancm-/- mice. Our findings document the first Mendelian phenotype due to a biallelic FANCM mutation.

Toxicity of different forms of graphene in a chicken embryo model.

In the present work, the toxicity of three forms of graphene: pristine graphene (pG), graphene oxide (GO), and reduced graphene oxide (rGO) was investigated using a chicken embryo model. Fertilized chicken eggs were divided into the control group and groups administered with pG, GO, and rGO, in concentrations of 50, 500, and 5000 µg/ml. The experimental solutions were injected in ovo into the eggs, and at day 18 of incubation, the embryo survival, body and organ weights, the ultrastructure of liver samples, and the concentration of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the livers were measured. Survival of embryos decreased significantly after treatment with all types of graphene, but not in a dose-dependent manner. The body weights were only slightly affected by the highest doses of graphene, while the organ weights were not different among treatment groups. In all experimental groups, atypical hepatocyte ultrastructure and mitochondrial damage were observed. The concentration of the marker of DNA damage 8-OHdG in the liver significantly decreased after pG and rGO treatments. Further in vivo studies with different animal models are necessary to clarify the level of toxicity of different types of graphene and to estimate the concentrations appropriate to evaluate their biomedical applications and environmental hazard.

Diamond Nanoparticles Modify Curcumin Activity: In Vitro Studies on Cancer and Normal Cells and In Ovo Studies on Chicken Embryo Model.

Curcumin has been studied broadly for its wide range of biological activities, including anticancer properties. The major problem with curcumin is its poor bioavailability, which can be improved by the addition of carriers, such as diamond nanoparticles (DN). They are carbon allotropes, and are therefore biocompatible and easily taken up by cells. DN are non-toxic and have antiangiogenic properties with potential applications in cancer therapy. Their large surface makes them promising compounds in a drug delivery system for bioactive agents, as DN create bio-complexes in a fast and simple process of self-organisation. We investigated the cytotoxicity of such bio-complexes against liver cancer cells and normal fibroblasts, revealing that conjugation of curcumin with DN significantly improves its activity. The experiment performed in a chicken embryo model demonstrated that neither curcumin nor DN nor bio-complexes affect embryo development, even though DN can form deposits in tissues. Preliminary results confirmed the applicability of DN as an efficient carrier of curcumin, which improves its performance against cancer cells in vitro, yet is not toxic to an organism, which makes the bio-complex a promising anticancer agent.

The effect of silver nanoparticles (AgNPs) on proliferation and apoptosis of in ovo cultured glioblastoma multiforme (GBM) cells.

Recently, it has been shown that silver nanoparticles (AgNPs) provide a unique approach to the treatment of tumors, especially those of neuroepithelial origin. Thus, the aim of this study was to evaluate the impact of AgNPs on proliferation and activation of the intrinsic apoptotic pathway of glioblastoma multiforme (GBM) cells cultured in an in ovo model. Human GBM cells, line U-87, were placed on chicken embryo chorioallantoic membrane. After 8 days, the tumors were divided into three groups: control (non-treated), treated with colloidal AgNPs (40 µg/ml), and placebo (tumors supplemented with vehicle only). At the end of the experiment, all tumors were isolated. Assessment of cell proliferation and cell apoptosis was estimated by histological, immunohistochemical, and Western blot analyses. The results show that AgNPs can influence GBM growth. AgNPs inhibit proliferation of GBM cells and seem to have proapoptotic properties. Although there were statistically significant differences between control and AgNP groups in the AI and the levels of active caspase 9 and active caspase 3, the level of these proteins in GBM cells treated with AgNPs seems to be on the border between the spontaneous apoptosis and the induced. Our results indicate that the antiproliferative properties of silver nanoparticles overwhelm proapoptotic ones. Further research focused on the cytotoxic effect of AgNPs on tumor and normal cells should be conducted.

Proliferative and apoptotic activity of glioblastoma multiforme cells cultured on in ovo model.

AIM: The influence of antitumor compounds on glioblastoma cell activity can be successfully investigated on an in ovo model. To consider an in ovo model as a reliable tool for estimation of anticancer drug efficacy, the biological activity of tumors growing in such conditions should be comparable to spontaneous cases. The aim of the present study was to evaluate the biological characteristics of glioblastoma multiforme (GBM) tumors - defined as their proliferative and apoptotic activities - growing on an in ovo model. MATERIALS AND METHODS: The GBM U-87 cell line was cultured on the chorioallantoic membrane of chicken eggs. After 12 days, cells were isolated and processed with H&E and immunohistochemical methods. The proliferative activity of GBM was established on the basis of mitotic and Ki-67(+) cells index. Apoptotic index was estimated by the Terminal Deoxynucleotidyl Transferase dUTP Nick-End Labeling (TUNEL) method. RESULTS: The biological activity of tumor tissue cultured in ovo corresponds to that of primary glioblastoma multiforme. CONCLUSION: GBM in in ovo model can be successfully applied in oncological studies.