Large Vacuoles Research Articles

Ciliogenesis and autophagy are coordinately regulated by EphA2 in the cornea to maintain proper epithelial architecture

PurposeTo understand the relationship between ciliogenesis and autophagy in the corneal epithelium. MethodssiRNAs for EphA2 or PLD1 were used to inhibit protein expression in vitro. Morpholino-anti-EphA2 was used to knockdown EphA2 in Xenopus skin. An EphA2 knockout mouse was used to conduct loss of function studies. Autophagic vacuoles were visualized by contrast light microscopy. Autophagy flux, was measured by LC3 turnover and p62 protein levels. Immunostaining and confocal microscopy were conducted to visualize cilia in cultured cells and in vivo. ResultsLoss of EphA2 (i) increased corneal epithelial thickness by elevating proliferative potential in wing cells, (ii) reduced the number of ciliated cells, (iii) increased large hollow vacuoles, that could be rescued by BafA1; (iv) inhibited autophagy flux and (v) increased GFP-LC3 puncta in the mouse corneal epithelium. This indicated a role for EphA2 in stratified epithelial assembly via regulation of proliferation as well as a positive role in both ciliogenesis and end-stage autophagy. Inhibition of PLD1, an EphA2 interacting protein that is a critical regulator of end-stage autophagy, reversed the accumulation of vacuoles, and the reduction in the number of ciliated cells due to EphA2 depletion, suggesting EphA2 regulation of both end-stage autophagy and ciliogenesis via PLD1. PLD1 mediated rescue of ciliogenesis by EphA2 depletion was blocked by BafA1, placing autophagy between EphA2 signaling and regulation of ciliogenesis. ConclusionOur findings demonstrate a novel role for EphA2 in regulating both autophagy and ciliogenesis, processes that are essential for proper corneal epithelial homeostasis.

Nuclear heterogeneity is prevalent in high-quality fractionated human sperm cells typically used for assisted conception.

What is the nuclear heterogeneity of high-density purified human spermatozoa typically used for IVF purposes. The data show that while density gradient separation has improved the overall sperm population, there is still a large degree of nuclear heterogeneity within these cells. Chromomycin A3 (CMA3) is an important DNA binding fluorochrome for the assessment of male-factor fertility. It is typically used to predict IVF outcomes on entire sperm ejaculates with very high receiver operating characteristic. Here we used CMA3 to characterise typical populations of human spermatozoa that would be used for IVF purposes after density gradient separation. We compared the intensity of CMA3 binding within high-dense sperm populations obtained from men. Binding heterogeneity was confirmed through fluorescence microscopy and FACS analysis independently. We also looked at CMA3 staining directly with head morphology in this sperm population. Finally, we looked at electron micrographs of nuclear heterogeneity (vacuoles, chromatin compaction) of spermatozoa following density gradient sorting of CMA3-stained cells. We used sperm donors who had fathered one or more children. Semen was collected after 2 days abstinence and purified over Percoll gradients. Only the high-quality spermatozoa, the same used for assisted conception, were then used. Cells were stained with CMA3 and sorted using FACS. Following this, electron micrographs were used to assess nuclear heterogeneity of CMA3-dependent sorted spermatozoa. CMA3 staining occurs within morphologically normal as well as abnormal spermatozoa. High-intensity CMA3-stained sperm possessed large vacuoles that were not seen in the low-CMA3 population. In addition, the high-CMA3 stained cells possess higher amounts of nuclear granulation. The present study only describes the issues within the chromatin of these cells and does not suggest an alternate selection technique. CMA3 is one of the better reported prognostic assays in predicting pregnancy outcomes, especially in cases where the male is at fault. However, it is clear that even in fractionated populations of human spermatozoa, there are sperm cells that are morphologically normal yet possess high levels of CMA3 staining and chromatin granulation. The implication of this is that the embryologist, whom selects on the basis of sperm morphology, may choose a cell with poor chromatin, which may lead to poor embryo outcomes. The project was funded by the National Health and Medical Research council, APP1118943. The authors have no conflict of interest to declare. N/A.

Thoughts on the evolution and ecological niche of diatoms

AbstractDiatoms are the most recent major algal lineage added to the geological record, appearing more than 200 million years ago. They are stramenopile protists resulting from a secondary endosymbiotic event that yielded the only photosynthetic protistan lineage expressing external siliceous cell wall structures called frustules. Many diatoms also have large internal vacuoles, and a common assumption in the literature is that success of the diatoms is largely attributable to these two morphological inventions: the frustule for defense and vacuole for luxury nutrient uptake. Here, we revisit the evolution of these inventions, propose sequential steps in frustule development, replace luxury nutrient uptake with predator defense and buoyancy control as the driver of vacuole expansion, and suggest that perhaps the greatest significance of the frustule for diatom evolution is the secondary consequence of enhancing sexual reproduction. In this synthesis, we emphasize a distinction between the “general” success of diatoms and the success of “bloom‐forming” species, as the physiological and morphological drivers of these successes differ. Importantly, the bloom‐forming species are responsible for the major role of diatoms in aquatic biogeochemical cycles. The bloom‐forming habit we ascribe to specific physiological attributes that, at their core, revolve around influencing the balance between diatom growth and losses to predators. We propose that these physiological adaptations are linked to size‐dependent maximum division rates in bloom‐forming diatoms, because of size scaling of predator–prey interactions. The existence of these bloom‐forming species yields an apparent allometric relationship that has previously been interpreted in terms of nutrient acquisition. Our analysis yields insights into species successions during blooms, considers the fundamental benefit of blooming (and subsequent sinking) from a reproductive standpoint, and provides some reinterpretation of diatoms success over geologic time and in the modern ocean.

126 EphA2 is a novel regulator of autolysosome recycling at end-stage autophagy and a key regulator in epidermal proliferation

Abnormal autophagy is observed in several skin diseases associated with hyperproliferation. We have shown that Beclin1 is increased in psoriatic lesions and when Beclin1 is conditionally knocked out in the epidermis, imiquimod(IMQ)-induced hyperproliferation is markedly attenuated. This indicates that autophagy plays a positive role in psoriasis and contributes to epidermal hyperproliferation in psoriatic lesions. However, how autophagy is regulated in the epidermis of psoriatic lesions is a knowledge gap that needs to be filled. We show that loss of EphA2, a receptor tyrosine kinase, reduces proliferation in mouse epidermis and human keratinocytes. Importantly, expression of EphA2 was shown to be increased in psoriatic lesions. We demonstrate that knockdown of EphA2 in HEKs results in an increase in: (i) large vacuoles; and (ii) LC3II protein, a marker for autophagosomes. To further explore this defect in autophagy, we measured autophagy flux by two well-established approaches: LC3II turnover and p62 levels, and found that loss of EphA2 reduced LC3II turnover in HEKs; and increased p62 in HEKs as well as in mouse epidermis, indicating an inhibition of autophagy flux. To determine the novel interactors of EphA2 in autophagy, we conducted BioID and co-immunoprecipitation assays and confirmed that among the novel binding partners of EphA2 was phospholipase D1 (PLD1). We have shown that PLD1 is a critical regulator of autolysosome recycling at end-stage autophagy. We have also demonstrated that the activity of dynamin1 (one of key players in autolysosome recycling at end-stage autophagy) and its association with autolysosomes is inhibited by PDL1/PKC pathway in HEKs. EphA2 affects autolysosome recycling via inhibiting PLD1/PKC signaling and thus ensuring dynamin1 activity and its association with autolysosome. Thus, we conclude that upregulation of EphA2 in the “activated” epidermis contributes to enhanced autophagy and consequently a hyperproliferative state.

Multiscale analysis of single and double maternal-zygotic Myh9 and Myh10 mutants during mouse preimplantation development.

During the first days of mammalian development, the embryo forms the blastocyst, the structure responsible for implanting the mammalian embryo. Consisting of an epithelium enveloping the pluripotent inner cell mass and a fluid-filled lumen, the blastocyst results from a series of cleavage divisions, morphogenetic movements, and lineage specification. Recent studies have identified the essential role of actomyosin contractility in driving cytokinesis, morphogenesis, and fate specification, leading to the formation of the blastocyst. However, the preimplantation development of contractility mutants has not been characterized. Here, we generated single and double maternal-zygotic mutants of non-muscle myosin II heavy chains (NMHCs) to characterize them with multiscale imaging. We found that Myh9 (NMHC II-A) is the major NMHC during preimplantation development as its maternal-zygotic loss causes failed cytokinesis, increased duration of the cell cycle, weaker embryo compaction, and reduced differentiation, whereas Myh10 (NMHC II-B) maternal-zygotic loss is much less severe. Double maternal-zygotic mutants for Myh9 and Myh10 show a much stronger phenotype, failing most of the attempts of cytokinesis. We found that morphogenesis and fate specification are affected but nevertheless carry on in a timely fashion, regardless of the impact of the mutations on cell number. Strikingly, even when all cell divisions fail, the resulting single-celled embryo can initiate trophectoderm differentiation and lumen formation by accumulating fluid in increasingly large vacuoles. Therefore, contractility mutants reveal that fluid accumulation is a cell-autonomous process and that the preimplantation program carries on independently of successful cell division.

The larval attachment organ of the tropical gar Atractosteus tropicus Gill, 1863 (Lepisosteiformes: Lepisosteidae).

Larval attachment organs (LAOs) are unicellular or multicellular organs that allow larvae to adhere to a substrate before yolk-sac absorption and the free-swimming stage. This study documents the LAO of tropical gar, Atractosteus tropicus, using a combination of scanning electron microscopy and light microscopy. It is shown that the LAO of A. tropicus is a super-organ surrounded by a wall and containing at its centre many smaller multicellular organ units, each comprised of attachment and support cells. Attachment cells are secretory and house large vacuoles filled with a glycoprotein. At hatching, the super-organ is well developed and occupies almost the entire anteroventral surface of the head. During subsequent development, the smaller individual units begin to regress, until at 6 days post-hatching the super-organ and its individual units are no longer visible.

Pendimethalin-based herbicide impairs cellular immune response and haemocyte morphology in a beneficial ground beetle

Herbicides have become the most commonly applied agrochemicals in agroecosystems. Thus, basic knowledge of their physiological effects on insects is needed, especially for understanding their impact on beneficial insect species. In this study, we evaluated the effect of a pendimethalin-based herbicide (PND) on the cellular immune response of the carabid beetle Harpalus (Pseudoophonus) rufipes (De Geer 1774) (Coleoptera, Carabidae), acting as biocontrol agent in agroecosystems. Total and differential haemocyte counts and phagocytosis assay, performed by injecting in vivo carboxylate-modified polystyrene latex beads, were measured in beetles exposed to a recommended field dose (4L per ha) of PND to evaluate the exposure effects over the time. The pattern of haemocyte subpopulations and the decrease of the phagocytic index after the exposure to PND suggested a lowering of P. rufipes ability to face an infection performing a cell-mediated response. PND was also found to cause cytotoxic effects on the haemocyte ultrastructure. Ultrastructural alterations such as irregular shape, large vacuolization of the cytoplasm, and condensation of marginated chromatin were recorded from 2d of exposure. The loss of RER, Golgi apparatus, mitochondria integrity and the swelling of the outer nuclear membrane found in some haemocytes suggested an interference of PND with the membrane permeability. Results indicated that the exposure to PND impairs the distribution, morphology and physiological functions of haemocytes causing a decrease of P. rufipes immunocompetence. Moreover, the sensitivity to herbicide exposure makes this species a suitable model and a useful bioindicator for monitoring exposure effects on non-target species. This study provides useful information to protect and preserve biodiversity of insects in agroecosystems.

Two new species of Unikaryon (Microsporidia) hyperparasitic in microphallid metacercariae (Digenea) from Florida intertidal crabs

The genus Unikaryon (Microsporidia) holds exclusively hyperparasites of Platyhelminthes. Four species of Unikaryon are presently known from trematodes infecting mollusks and fish, and one from a cestode infecting a fish. Here we report two species of Unikaryon from microphallid trematode metacercariae parasitizing the brachyuran crabs, Panopeus herbstii and Pachygrapsus transversus, collected from intertidal habitats in Florida. The first microsporidium, which we assign here to a new species, Unikaryon panopei sp. n., was isolated from Microphallus sp. encysted in Panopeus herbstii from Tampa Bay. The specific designation for the second Unikaryon sp. (Unikaryon sp. 2), which occurred in metacercaria of Diacetabulum sp. found in P. transversus from the Florida Keys, is pending due to the lack of SSrDNA sequence data. Light and electron microscopy demonstrates that both species display characteristics of the genus Unikaryon including the arrangement of spores in sets of two, large posterior vacuole, and eccentric position of the polar filament. Spores of Unikaryon panopei sp. n., unlike those of Unikaryon sp. 2, assemble in large membrane-bound masses containing hundreds of organisms, and display a larger number of polar filament coils – 7–8, compared to 4–5 in Unikaryon sp. 2 The SSUrDNA-inferred phylogenetic analysis places Unikaryon panopei in one clade with Unikaryon legeri, the only other molecularly characterized member of the genus, with 94% of SSUrDNA similarity. These findings increase the number of species parasitizing trematodes and broaden the host range of Unikaryon spp.

Insulinase-like Protease 1 Contributes to Macrogamont Formation in Cryptosporidium parvum.

The apicomplexan parasite Cryptosporidium parvum contains an expanded family of 22 insulinase-like proteases (INS), a feature that contrasts with their otherwise streamlined genome. Here, we examined the function of INS1, which is most similar to the human insulinase protease that cleaves a variety of small peptide substrates. INS1 is an M16A clan member and contains a signal peptide, an N-terminal domain with the HXXEH active site, followed by three inactive domains. Unlike previously studied C. parvum INS proteins that are expressed in sporozoites and during merogony, INS1 was expressed exclusively in macrogamonts, where it was localized in small cytoplasmic vesicles. Although INS1 did not colocalize with the oocyst wall protein recognized by the antibody OW50, immune-electron microscopy indicated that INS1 resides in small vesicles in the secretory system. Notably, these small INS1-positive vesicles were often in close proximity to large OW50-positive vacuoles resembling wall-forming bodies, which contain precursors for oocyst wall formation. Genetic deletion of INS1, or replacement with an active-site mutant, resulted in lower formation of macrogamonts in vitro and reduced oocyst shedding in vivo Our findings reveal that INS1 functions in the formation or maturation of macrogamonts and that its loss results in attenuated virulence in immunocompromised mice.IMPORTANCE Cryptosporidiosis is a debilitating diarrheal disease in young children in developing countries. The absence of effective treatments or vaccines makes this infection very difficult to manage in susceptible populations. Although the oral dose of oocysts needed to cause infection is low, infected individuals shed very high numbers of oocysts, readily contaminating the environment. Our studies demonstrate that the protease INS1 is important for formation of female sexual stages and that in its absence, parasites produce fewer oocysts and are attenuated in immunocompromised mice. These findings suggest that mutants lacking INS1, or related proteases, are useful for further characterizing the pathway that leads to macrogamont maturation and oocyst wall formation.

Potential Antiviral Target for SARS-CoV-2: A Key Early Responsive Kinase during Viral Entry

Currently, there is no effective antiviral medication for coronavirus disease 2019 (COVID-19) and the knowledge on the potential therapeutic target is in great need. Guided by a time-course transmi...

Characteristic vacuolisation of granulocytic and erythroid precursors associated with VEXAS syndrome.

A 78-year-old man with Sweet’s syndrome was referred for progressive anaemia. The full blood count showed macrocytic non-regenerative anaemia (haemoglobin concentration 82 g/l, mean cell volume 107 fl, reticulocytes 34 × 109/l) without neutropenia or thrombocytopenia. Bone marrow examination (images, ×100 objective) showed isolated dyserythropoiesis with basophilic stippling (top left), nuclear budding (bottom left), giant erythroblasts with nuclear:cytoplasmic asynchrony (bottom left) and 22% ring sideroblasts (Perls stain, top centre), without blast excess. This result was indicative of a myelodysplastic syndrome with ring sideroblasts and single lineage dysplasia (MDS-RS-SLD). Moreover, large vacuoles were observed in 20% of granulocytic (top right and bottom centre) and 10% of erythroid precursors (bottom right) but not in lymphocytes. Next-generation sequencing (NGS) did not reveal any classical MDS mutations but identified a UBA1 (ubiquitin-activating enzyme 1) M41L mutation (variant allele frequency 71%).

Effects of Tyrosine and Tryptophan in Rats with Diet-Induced Obesity.

Amino acids tyrosine (Tyr) and tryptophan (Trp) play a significant role in the regulation of energy metabolism, locomotor activity, and eating behavior. We studied the possibility of modulating these processes in obesity by increasing the pool of Tyr and Trp in the experimental diet. As a model of obesity, we used Wistar rats fed a diet with an excess specific energy value (HFCD) for 64 days. Trp led to a normalization of the rats’ body weight almost to the control level, but increased anxiety-like behavior and decreased long-term memory. The consumption of amino acids resulted in increased grip strength and impairment of short-term memory. The locomotor activity of animals decreased with age as a result of Tyr consumption, while Trp, on the contrary, prevented this. The Tyr supplementation led to the normalization of triglycerides and LDL. In the spleen cell lysates, amino acids suppressed the production of proinflammatory cytokines. The liver tissue morphology showed that the consumption of Tyr noticeably weakened the signs of fatty degeneration. The addition of Trp, on the contrary, led to an unfavorable effect, consisting of the appearance of a high number of large rounded fatty vacuoles. The data obtained indicate a more pronounced anti-inflammatory effect of Tyr as compared to Trp.

Extracts Prepared from a Canadian Toxic Plant Induce Light-Dependent Perinuclear Vacuoles in Human Cells.

We are investigating plant species from the Canadian prairie ecological zone by phenotypic cell assays to discover toxins of biological interest. We provide the first report of the effects of extracts prepared from the shrub Symphoricarpos occidentalis in several human cell lines. S. occidentalis (Caprifoliaceae) extracts are cytotoxic, and, strikingly, treated cells undergo light-dependent vacuolation near the nucleus. The range of irradiation is present in standard ambient light and lies in the visible range (400-700 nm). Vacuolization in treated cells can be induced with specific wavelengths of 408 or 660 nm at 1 J/cm2 energies. Vacuolated cells show a striking phenotype of a large perinuclear vacuole (nuclear associated vacuole, NAV) that is distinct from vesicles observed by treatment with an autophagy-inducing agent. Treatment with S. occidentalis extracts and light induces an intense lamin A/C signal at the junction of a nuclear vacuole and the nucleus. Further study of S. occidentalis extracts and vacuolation provide chemical tools that may contribute to the understanding of nuclear envelope organization and human cell biology.Key ContributionWe provide the first description of the biological effects upon human cells of extracts from the toxic plant, Symphoricarpos occidentalis. Treated cells acquire striking nuclear associated vacuoles (NAVs), rarely observed in animal cell biology.

Gellan gum hydrogels cross-linked with carbodiimide stimulates vacuolation of human tooth-derived stem cells in vitro

The natural polysaccharides are promising compounds for applications in regenerative medicine. Gellan gum (GG) is the bacteria-derived polysaccharide widely used in food industry. Simple modifications of its chemical properties make GG superior for the development of biocompatible hydrogels. Beside reversible cationic integration of GG chains, more efficient binding is accomplished with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). However, the side-products of polymer cross-linking might affect viability and differentiation of stem cells introduced into the hydrogels. We found that O-acylisourea (EDU) stimulates autophagy-based vacuolation in both periodontal ligament and dental pulp stem cells. 24-h treatment of cells with GG extracts cross-linked with 15 mM EDC developed large cytoplasmic vacuoles. Freshly prepared EDU (2–6 mM) but not 15 mM EDC solutions initiated vacuole development with concomitant reduction of cell viability/metabolism. Most of the vacuoles stained with acridine orange displayed highly acidic environment further confirmed by flow cytometric analysis. Western blot of the LC3 autophagy marker followed by a transmission electron microscopy indicated the process is autophagy-dependent. We propose that the high reactivity of EDU with intracellular components initiates autophagy, although the targets of EDU remain unknown. Nevertheless, a burst release of EDU from GG hydrogels might modulate negatively cellular processes and final effectiveness of tissue regeneration.

Fabrication of solid CH-CD multilayer microspheres for inertial confinement fusion

Deuterated polymer microspheres can be used as a neutron source in conjunction with lasers because thermonuclear fusion neutrons can be produced efficiently by collisions of the resulting energetic deuterium ions. A new type of solid deuterated polymer microsphere with a carbon hydrogen–carbon deuterium (CH-CD) multilayer has been designed for preparing the target for inertial confinement fusion (ICF) experiments. To fabricate these solid CH-CD multilayer microspheres, CH beads are first fabricated by a microfluidic technique, and the CD coating layer is prepared by a plasma polymerization method. Both polystyrene (PS) and poly(α-methylstyrene) (PAMS) are used as the material sources for the CH beads. The effects of the PS and PAMS materials on the quality of the solid CH beads and the resulting CH-CD multilayer polymer microspheres are investigated. The solid PS beads have better sphericity and a smoother surface, but large vacuoles are observed in solid PS-CD multilayer microspheres owing to the presence of residual fluorobenzene in the beads and a glass transition temperature of the solid PS beads that is lower than the temperature of plasma polymerization. Therefore, solid PAMS beads are more suitable as a mandrel for fabricating solid CH-CD multilayer polymer microspheres. Solid CH-CD multilayer microspheres with specified size have been successfully prepared by controlling the droplet size and the CD deposition rate and deposition time. Compared with the design value, the diameter deviation of the inner CH beads and the thickness deviation of the CD layer can be controlled within 20 µm and 2 µm, respectively. Thus, an approach has been developed to fabricate solid CH-CD multilayer microspheres that meet the physical design requirements for ICF.

Localization of Claudin-3 and Claudin-4 within the Small Intestine of newborn piglets.

Piglets must acquire passive immunity through colostrum within hours after birth to survive. How colostral macromolecules traverse the small intestinal epithelium may include nonselective pinocytosis and paracellular transport through tight junction proteins located between epithelial cells. Claudin proteins‐3 and ‐4 contribute to the epithelial tight junctions (TJs) on the apical aspect of lateral surfaces of intestinal epithelial cells (IECs) where they help regulate ion and macromolecule movement across the intestinal epithelium. Throughout the small intestine of newborn piglets, Claudin‐3 was localized to the lateral and basolateral surface of intestinal epithelial cells as well as the membrane of large vacuoles. In the duodenum and jejunum, Claudin‐4 was localized to the apical surface independent of tight junction regions. In the ileum, Claudin‐4 was localized to the lateral and basolateral surfaces indicating region‐specific differences and noncanonical patterns of Claudin‐4 localization independent of tight junction regions. Understanding the timing of changes in surface localization of Claudin‐3 and Claudin‐4 and how they may coincide with changes in small intestinal permeability may help develop new protective strategies against infectious diseases within newborn piglets.

Ultra-structural analysis and morphological changes during the differentiation of trophozoite to cyst in Entamoeba invadens

Entamoeba histolytica, a pathogenic parasite, is the causative organism of amoebiasis and uses human colon to complete its life cycle. It destroys intestinal tissue leading to invasive disease. Since it does not form cyst in culture medium, a reptilian parasite Entamoeba invadens serves as the model system to study encystation. Detailed investigation on the mechanism of cyst formation, information on ultra-structural changes and cyst wall formation during encystation are still lacking in E. invadens. Here, we used electron microscopy to study the ultrastructural changes during cyst formation and showed that the increase in heterochromatin patches and deformation of nuclear shape were early events in encystation. These changes peaked at ∼20 h post induction, and normal nuclear morphology was restored by 72 h. Two types of cellular structures were visible by 16 h. One was densely stained and consisted of the cytoplasmic mass with clearly visible nucleus. The other consisted of membranous shells with large vacuoles and scant cytoplasm. The former structure developed into the mature cyst while the latter structure was lost after 20 h, This study of ultra-structural changes during encystation in E. invadens opens up the possibilities for further investigation into the mechanisms involved in this novel process.

Effects of organophosphate insecticide, sumithion on histopathology of common carp (Cyprinus carpio) in the natural pond condition

Sumithion is an organophosphorus insecticide, which is widely used in aquaculture ponds for eradication of aquatic insect (mainly tiger bug) prior to release of larvae. This study was conducted to evaluate the effects of an organophosphorus pesticide, sumithion on histoarchitecture of liver and kidney in common carp (Cyprinus carpio) during the period from July to December 2016. It was carried out with four treatments, each with two replications. i.e., ponds with no sumithion (T0), with 0.025ppm sumithion (T1), 0.050 ppm sumithion (T2) and 0.100 ppm sumithion (T3). The water quality parameters, such as pH, dissolved oxygen, total alkalinity, free CO2, nitrate-nitrogen (NO3-N), phosphate-phosphorus (PO4-P) were not affected by sub lethal doses of sumithion but the values were fluctuated significantly in most of the cases between the ponds throughout the study period. In case of histoarchitecture of liver and kidney, normal structure of liver and kidney cells were observed in the controlled and treated fish. Through the histological analysis of liver, small vacuole, enlarge lumen space of hepatopancreas and disrupted hepatopancreas were found in T1. Disrupted hepatopancreas, increasement intracellular space, regeneration of hepatic cell and hemorrhage were observed in T2. Moreover, enlarge lumen space of hepatopancreas, degenerated hepatic cell, disrupted hepatopancreas were observed in T3. After the histological analysis of kidney, degenerated renal corpuscle, enlargement of blood vessel, disrupted hematopoetic cell were observed in T1. Ruptured collecting duct, large vacuole, enlarge intracellular space were observed in T2. Furthermore, enlarge bowman’s space, degenerated hematopoetic cell hemorrhage and ruptured distal tubule disrupted, enlarge intracellular space and necrosis were observed in T3. The present investigation sufficiently emphasizes that sumithion has adverse effects on the major organs like liver and kidney.
 Int. J. Agril. Res. Innov. Tech. 10(2): 66-75, December 2020

5-Fluorouracil disrupts ovarian preantral follicles in young C57BL6J mice.

5-Fluorouracil (5-FU), an anti-cancer drug, has been used for hepatoblastoma (HB) chemotherapy in children, who mayhave impaired ovarian follicle pool reserve with lasting effectsto reproduction. Therefore, this study aimed to investigate 5-FU effects on survival, growth, and morphology of ovarian preantral follicles from C57BL6J young mice. Experiments were carried-out both in vivo and in vitro. Mice were treated with 5-FU injection (450mg/kg i.p) or saline and sacrificed 3days after to obtain ovaries for histology and molecular biology. Ovaries for in vitro studies were obtained from unchallenged mice and cultured under basic culture medium (BCM) or BCM plus 5-FU (9.2, 46.1, 92.2mM). Preantral follicles were classified according to developmental stages, and as normal or degenerated. To assess cell viability, caspase-3 immunostaining was performed. Transcriptional levels for apoptosis (Bax, Bcl2, p53, Bax/Bcl2) and Wnt pathway genes (Wnt2 and Wnt4) were also analyzed. Ultrastructural analyses were carried-out on non-cultured ovaries. In addition, β-catenin immunofluorescence was assessed in mouse ovaries. The percentage of all-types normal follicles was significantly lower after 5-FU challenge. A total loss of secondary normal follicles was found in the 5-FU group. The highest 5-FU concentrations reduced the percentage of cultured normal primordial follicles. Large vacuoles were seen in granulosa cells and ooplasm of preantral follicles by electron microscopy. A significantly higher gene expression for Bax and Bax/Bcl2 ratio was seen after 5-FU treatment. A marked reduction in β-catenin immunolabeling was seen in 5-FU-challenged preantral follicles. In the in vitro experiments, apoptotic and Wnt gene transcriptions were significantly altered. Altogether, our findings suggest that 5-FU can deleteriously affect the ovarian follicle reserve by reducing preantral follicles survival.

Giardia intestinalis can interact, change its shape and internalize large particles and microorganisms.

Giardia intestinalis is a parasitic protozoan that inhabits its vertebrate hosts' upper small intestine and is the most common cause of waterborne diarrhoea worldwide. Giardia trophozoites present few organelles, and among them, they possess peripheral vesicles (PVs), which are considered an endosomal-lysosomal system. All experimental procedures carried out until now indicate that Giardia ingests macromolecules by fluid-phase and receptor-mediated endocytic pathways. Still, there is no description concerning the interaction and ingestion of large materials. Here, we tested Giardia's capacity to interact with large particles; once, in vivo, it inhabits an environment with a microbiota. We tested protozoan interaction with yeasts, bacteria, latex beads, ferritin and albumin, in different times of interaction and used several microscopy techniques (light microscopy, scanning electron microscopy and transmission electron microscopy) to follow their fate. Giardia interacted with all of the materials we tested. Projections of the plasma membrane similar to pseudopods were seen. As albumin, small markers were found in the PVs while the larger materials were not seen there. Large vacuoles containing large latex beads were detected intracellularly. Thus, we observed that: (1) Giardia interacts with large materials; (2) Giardia can display an amoeboid shape and exhibit membrane projections when in contact with microorganisms and large inorganic materials; (3) the region of the exit of the ventral flagella is very active when in contact with large materials, although all cell surface also present activity in the interactions; (4) intracellular vacuoles, which are not the PVs, present ingested large beads.