Effect of quercetin or butylated hydroxytoluene on cooled or frozen-thawed ram sperm quality

Cooling and freezing processes cause physical and chemical damage to sperm by cold shock and oxidative stress. This study aimed to evaluate the effect of two antioxidants on sperm parameters of cooled and frozen-thawed ram semen diluted in an egg yolk-based extender. Semen was collected from 30 rams and processed in two consecutive experiments to test the inclusion of different concentrations of quercetin and butylated hydroxytoluene (BHT) in an egg yolk-based semen extender. Dimethyl sulfoxide (DMSO) was added as a solvent to the semen extender in a ratio of 1 mL DMSO for 90 mg of quercetin and 1 mL DMSO for 880 mg of BHT. After collection, semen was diluted at 200 × 106 motile sperm/mL (control) and split into different groups in each experiment. In experiment 1, semen was diluted with the extender containing quercetin (Q5, 5 μg/mL; Q10, 10 μg/mL; Q15, 15 μg/mL) or DMSO alone (DMSO1, 0.055 μL DMSO per mL; DMSO2, 0.165 μL DMSO per mL). In experiment 2, semen was diluted with the extender with BHT (BHT1, 0.5 μg/mL; BHT2, 1 μg/mL; BHT3, 1.5 μg/mL) or DMSO alone (DMSO3, 0.375 μL DMSO per mL; DMSO4, 1.125 μL DMSO per mL). After dilution, the semen was divided into two aliquots. Treated ram sperm samples were also subjected to different storage methods. The first set of samples was cooled at 5 °C for 24 h, whereas the second set of samples was frozen-thawed. Sperm motility parameters and plasma membrane integrity (PMI) were evaluated immediately after dilution (0h) and 24 h after cooling and in the frozen-thawed samples via computer-assisted sperm analysis and epifluorescence microscopy, respectively. The inclusion of quercetin or BHT did not affect sperm motility parameters or PMI of fresh, cooled, or frozen-thawed sperm in this study (P < 0.05). However, further studies are needed to test the effects of these antioxidants on the fertility of cryopreserved ram semen.

The Anthelmintic Quassinoids Ailanthone and Bruceine a Induce Infertility in the Model Organism Caenorhabditis elegans by an Apoptosis-like Mechanism Induced in Gonadal and Spermathecal Tissues

In continuation of the search for new anthelmintic natural products, the study at hand investigated the nematicidal effects of the two naturally occurring quassinoids ailanthone and bruceine A against the reproductive system of the model nematode Caenorhabditis elegans to pinpoint their anthelmintic mode of action by the application of various microscopic techniques. Differential Interference Contrast (DIC) and the epifluorescence microscopy experiments used in the presented study indicated the genotoxic effects of the tested quassinoids (c ailanthone = 50 µM, c bruceine A = 100 µM) against the nuclei of the investigated gonadal and spermathecal tissues, leaving other morphological key features such as enterocytes or body wall muscle cells unimpaired. In order to gain nanoscopic insight into the morphology of the gonads as well as the considerably smaller spermathecae of C. elegans, an innovative protocol of polyethylene glycol embedding, ultra-sectioning, acridine orange staining, tissue identification by epifluorescence, and subsequent AFM-based ultrastructural data acquisition was applied. This sequence allowed the facile and fast assessment of the impact of quassinoid treatment not only on the gonadal but also on the considerably smaller spermathecal tissues of C. elegans. These first-time ultrastructural investigations on C. elegans gonads and spermathecae by AFM led to the identification of specific quassinoid-induced alterations to the nuclei of the reproductive tissues (e.g., highly condensed chromatin, impaired nuclear membrane morphology, as well as altered nucleolus morphology), altogether implying an apoptosis-like effect of ailanthone and bruceine A on the reproductive tissues of C. elegans.

Cell-wall fluorescence highlights the phases of xylogenesis

The monitoring of xylogenesis makes it possible to follow tree growth responses to stress factors in real-time, by observing the course of wood cell division and differentiation. Proper microscopy techniques are of key importance to exactly identify the xylem cells during the different phases of differentiation. We aimed to apply epifluorescence microscopy to follow the lignification process during the different phases of xylogenesis in Mediterranean softwood and hardwood. Microcores from trees of Pinus halepensis Mill. and Arbutus unedo L. were collected at a site in southern Italy, during the period June-December. Fluorescence imaging of sections stained with a water solution of safranin and Astra blue clearly highlighted the contrast between lignified and un-lignified tissue. The proposed methodology is useful to quickly and unambiguously detect the different stages of cell differentiation, as well as the progress in the lignification process. Moreover, it proved to be easily applied to demanding wood materials, such as Mediterranean woods and can be helpful to better track stress responses and the development of anomalies during wood formation, such as intra-annual density fluctuations.

NMDA receptor expression during cell transformation process at early stages of liver cancer in rodent models

Hepatocellular carcinoma (HCC) is the most common primary liver cancer, which is not sensitive to radiotherapy and chemotherapy and very often experiences postoperative relapse. In this regard, effective screening of liver cancer is considered as the most important and urgent task. The aim of our study was to determine whether N-methyl-D-aspartate receptor (NMDAR) and, in particular, its subunits, can serve as biomarkers to distinguish the precancerous liver at early stages of liver fibrosis. We assessed the development of HCC after 10, 15 and 22 weeks using a HCC rat model. The expression of NMDAR subunits was monitored at different stages of HCC by means of immunohistochemistry combined with epifluorescence microscopy imaging, Western blotting and direct bisulfite sequencing. NMDAR subunits were not found in healthy liver tissues. In contrast, NMDAR subunits, in particular NR1 and NR2B, appeared at the stage of severe liver fibrosis (precancerous liver disease) in rats and were expressed during the development of HCC in rats and mice. Using the direct bisulfite sequencing, we detected that increased expression of NMDAR directly correlated with the demethylation of CpG islands in the promoter region of genes encoding receptor subunits. The obtained results confirmed that NMDAR subunits can serve as new biomarkers of precancerous liver disease, severe fibrosis, and its progression towards HCC.

High-Pressure-Induced Sublethal Injuries of Food Pathogens—Microscopic Assessment

High Hydrostatic Pressure (HHP) technology is considered an alternative method of food preservation. Nevertheless, the current dogma is that HHP might be insufficient to preserve food lastingly against some pathogens. Incompletely damaged cells can resuscitate under favorable conditions, and they may proliferate in food during storage. This study was undertaken to characterize the extent of sublethal injuries induced by HHP (300–500 MPa) on Escherichia coli and Listeria inncua strains. The morphological changes were evaluated using microscopy methods such as Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), and Epifluorescence Microscopy (EFM). The overall assessment of the physiological state of tested bacteria through TEM and SEM showed that the action of pressure on the structure of the bacterial membrane was almost minor or unnoticeable, beyond the L. innocua wild-type strain. However, alterations were observed in subcellular structures such as the cytoplasm and nucleoid for both L. innocua and E. coli strains. More significant changes after the HHP of internal structures were reported in the case of wild-type strains isolated from raw juice. Extreme condensation of the cytoplasm was observed, while the outline of cells was intact. The percentage ratio between alive and injured cells in the population was assessed by fluorescent microscopy. The results of HHP-treated samples showed a heterogeneous population, and red cell aggregates were observed. The percentage ratio of live and dead cells (L/D) in the L. innocua collection strain population was higher than in the case of the wild-type strain (69%/31% and 55%/45%, respectively). In turn, E. coli populations were characterized with a similar L/D ratio. Half of the cells in the populations were distinguished as visibly fluorescing red. The results obtained in this study confirmed sublethal HHP reaction on pathogens cells.

Bacterial Abundance, Diversity and Activity During Long-Term Colonization of Non-biodegradable and Biodegradable Plastics in Seawater

The microorganisms living on plastics called “plastisphere” have been classically described as very abundant, highly diverse, and very specific when compared to the surrounding environments, but their potential ability to biodegrade various plastic types in natural conditions have been poorly investigated. Here, we follow the successive phases of biofilm development and maturation after long-term immersion in seawater (7 months) on conventional [fossil-based polyethylene (PE) and polystyrene (PS)] and biodegradable plastics [biobased polylactic acid (PLA) and polyhydroxybutyrate-co-hydroxyvalerate (PHBV), or fossil-based polycaprolactone (PCL)], as well as on artificially aged or non-aged PE without or with prooxidant additives [oxobiodegradable (OXO)]. First, we confirmed that the classical primo-colonization and growth phases of the biofilms that occurred during the first 10 days of immersion in seawater were more or less independent of the plastic type. After only 1 month, we found congruent signs of biodegradation for some bio-based and also fossil-based materials. A continuous growth of the biofilm during the 7 months of observation (measured by epifluorescence microscopy and flow cytometry) was found on PHBV, PCL, and artificially aged OXO, together with a continuous increase in intracellular (3H-leucine incorporation) and extracellular activities (lipase, aminopeptidase, and β-glucosidase) as well as subsequent changes in biofilm diversity that became specific to each polymer type (16S rRNA metabarcoding). No sign of biodegradation was visible for PE, PS, and PLA under our experimental conditions. We also provide a list of operational taxonomic units (OTUs) potentially involved in the biodegradation of these polymers under natural seawater conditions, such as Pseudohongiella sp. and Marinobacter sp. on PCL, Marinicella litoralis and Celeribacter sp. on PHBV, or Myxococcales on artificially aged OXO. This study opens new routes for a deeper understanding of the polymers’ biodegradability in seawaters, especially when considering an alternative to conventional fossil-based plastics.

Coral Reef Viruses in Kane'ohe Bay, Hawai'i: Abundance, Diversity & Environmental Drivers

<p>Viruses are a ubiquitous component of coral reef ecosystems, with several viral types, from at least seven prokaryotic and 20 eukaryotic virus families currently characterised from the surface mucopolysaccharide layer (SML), coral tissue and the water column. However, little is known about the ecology and function of these viruses. For example, what are the environmental drivers of viral abundance and diversity on coral reefs? In this study, the abundance and distribution of virus-like particles (VLPs) associated with the SML and reef water of the coral Montipora capitata were determined using epifluorescence microscopy, while transmission electron microscopy was employed to determine the morphological diversity of VLPs. Sampling was conducted across the Coconut Island Marine Reserve (CIMR) reef system, Kane’ohe Bay, O’ahu, Hawai’i. Viral abundance was correlated with select environmental drivers and prokaryote abundance, while non-metric multidimensional scaling was used to determine the key environmental drivers of the viral community assemblage. The water column contained high concentrations of VLPs (5.98 × 107 ml-1) and prokaryotes (3.11 × 106 ml-1), consistent with the considerable anthropogenic impacts at this location. In comparison, the SML contained lower concentrations of VLPs (2.61 × 107 ml-1) and prokaryotes (2.08 × 106 ml-1); of note, the densities of viruses and prokaryotes in the SML were strongly coupled while those in the reef water were not. VLP density in the water column varied spatially across the reef, with the most sheltered site and the only one not situated on the reef crest having a greater VLP density than the other sites. Temporal variations in the density of microbes (i.e. viruses and prokaryotes) in the reef water were pronounced, while in the SML microbial densities remained constant. However, no specific environmental drivers of this variability could be identified. In contrast, temperature and water quality were correlated with shifts in the morphological diversity of VLPs across the reef. Small (< 50 nm) polyhedral/spherical VLPs were dominant, and were positively correlated to chlorophyll-a concentration when in the SML. In this same habitat, Fuselloviridae-like VLPs, filamentous VLPs and bead-shaped VLPs were positively correlated to temperature. In the reef water a different pattern was apparent: large (> 100 nm) Podoviridae-like VLPs and elongate Myoviridae-like VLPs, as well as lemon-shaped VLPs of both size classes showed positive associations with turbidity, while large filamentous VLPs, Geminiviridae-like VLPs and rod-shaped VLPs were positively associated with temperature. These results demonstrate that the viral community of Coconut Island’s reef is highly diverse, and subject to spatial and temporal change, especially in the water column. However, while the environmental drivers of viral diversity were partly elucidated, we are still a long way from understanding the drivers of viral abundance. More detailed study, both spatially and temporally, of the CIMR environment is required, as is comprehensive molecular analysis of the viral community of Kane’ohe Bay. Only then can we begin to understand the importance of viruses to the health and function of this, and other reef sites.</p>

Coral Reef Viruses in Kane'ohe Bay, Hawai'i: Abundance, Diversity & Environmental Drivers

<p>Viruses are a ubiquitous component of coral reef ecosystems, with several viral types, from at least seven prokaryotic and 20 eukaryotic virus families currently characterised from the surface mucopolysaccharide layer (SML), coral tissue and the water column. However, little is known about the ecology and function of these viruses. For example, what are the environmental drivers of viral abundance and diversity on coral reefs? In this study, the abundance and distribution of virus-like particles (VLPs) associated with the SML and reef water of the coral Montipora capitata were determined using epifluorescence microscopy, while transmission electron microscopy was employed to determine the morphological diversity of VLPs. Sampling was conducted across the Coconut Island Marine Reserve (CIMR) reef system, Kane’ohe Bay, O’ahu, Hawai’i. Viral abundance was correlated with select environmental drivers and prokaryote abundance, while non-metric multidimensional scaling was used to determine the key environmental drivers of the viral community assemblage. The water column contained high concentrations of VLPs (5.98 × 107 ml-1) and prokaryotes (3.11 × 106 ml-1), consistent with the considerable anthropogenic impacts at this location. In comparison, the SML contained lower concentrations of VLPs (2.61 × 107 ml-1) and prokaryotes (2.08 × 106 ml-1); of note, the densities of viruses and prokaryotes in the SML were strongly coupled while those in the reef water were not. VLP density in the water column varied spatially across the reef, with the most sheltered site and the only one not situated on the reef crest having a greater VLP density than the other sites. Temporal variations in the density of microbes (i.e. viruses and prokaryotes) in the reef water were pronounced, while in the SML microbial densities remained constant. However, no specific environmental drivers of this variability could be identified. In contrast, temperature and water quality were correlated with shifts in the morphological diversity of VLPs across the reef. Small (< 50 nm) polyhedral/spherical VLPs were dominant, and were positively correlated to chlorophyll-a concentration when in the SML. In this same habitat, Fuselloviridae-like VLPs, filamentous VLPs and bead-shaped VLPs were positively correlated to temperature. In the reef water a different pattern was apparent: large (> 100 nm) Podoviridae-like VLPs and elongate Myoviridae-like VLPs, as well as lemon-shaped VLPs of both size classes showed positive associations with turbidity, while large filamentous VLPs, Geminiviridae-like VLPs and rod-shaped VLPs were positively associated with temperature. These results demonstrate that the viral community of Coconut Island’s reef is highly diverse, and subject to spatial and temporal change, especially in the water column. However, while the environmental drivers of viral diversity were partly elucidated, we are still a long way from understanding the drivers of viral abundance. More detailed study, both spatially and temporally, of the CIMR environment is required, as is comprehensive molecular analysis of the viral community of Kane’ohe Bay. Only then can we begin to understand the importance of viruses to the health and function of this, and other reef sites.</p>

Coral Reef Viruses in Kane'ohe Bay, Hawai'i: Abundance, Diversity & Environmental Drivers

<p>Viruses are a ubiquitous component of coral reef ecosystems, with several viral types, from at least seven prokaryotic and 20 eukaryotic virus families currently characterised from the surface mucopolysaccharide layer (SML), coral tissue and the water column. However, little is known about the ecology and function of these viruses. For example, what are the environmental drivers of viral abundance and diversity on coral reefs? In this study, the abundance and distribution of virus-like particles (VLPs) associated with the SML and reef water of the coral Montipora capitata were determined using epifluorescence microscopy, while transmission electron microscopy was employed to determine the morphological diversity of VLPs. Sampling was conducted across the Coconut Island Marine Reserve (CIMR) reef system, Kane’ohe Bay, O’ahu, Hawai’i. Viral abundance was correlated with select environmental drivers and prokaryote abundance, while non-metric multidimensional scaling was used to determine the key environmental drivers of the viral community assemblage. The water column contained high concentrations of VLPs (5.98 × 107 ml-1) and prokaryotes (3.11 × 106 ml-1), consistent with the considerable anthropogenic impacts at this location. In comparison, the SML contained lower concentrations of VLPs (2.61 × 107 ml-1) and prokaryotes (2.08 × 106 ml-1); of note, the densities of viruses and prokaryotes in the SML were strongly coupled while those in the reef water were not. VLP density in the water column varied spatially across the reef, with the most sheltered site and the only one not situated on the reef crest having a greater VLP density than the other sites. Temporal variations in the density of microbes (i.e. viruses and prokaryotes) in the reef water were pronounced, while in the SML microbial densities remained constant. However, no specific environmental drivers of this variability could be identified. In contrast, temperature and water quality were correlated with shifts in the morphological diversity of VLPs across the reef. Small (< 50 nm) polyhedral/spherical VLPs were dominant, and were positively correlated to chlorophyll-a concentration when in the SML. In this same habitat, Fuselloviridae-like VLPs, filamentous VLPs and bead-shaped VLPs were positively correlated to temperature. In the reef water a different pattern was apparent: large (> 100 nm) Podoviridae-like VLPs and elongate Myoviridae-like VLPs, as well as lemon-shaped VLPs of both size classes showed positive associations with turbidity, while large filamentous VLPs, Geminiviridae-like VLPs and rod-shaped VLPs were positively associated with temperature. These results demonstrate that the viral community of Coconut Island’s reef is highly diverse, and subject to spatial and temporal change, especially in the water column. However, while the environmental drivers of viral diversity were partly elucidated, we are still a long way from understanding the drivers of viral abundance. More detailed study, both spatially and temporally, of the CIMR environment is required, as is comprehensive molecular analysis of the viral community of Kane’ohe Bay. Only then can we begin to understand the importance of viruses to the health and function of this, and other reef sites.</p>

Coral Reef Viruses in Kane'ohe Bay, Hawai'i: Abundance, Diversity & Environmental Drivers

<p>Viruses are a ubiquitous component of coral reef ecosystems, with several viral types, from at least seven prokaryotic and 20 eukaryotic virus families currently characterised from the surface mucopolysaccharide layer (SML), coral tissue and the water column. However, little is known about the ecology and function of these viruses. For example, what are the environmental drivers of viral abundance and diversity on coral reefs? In this study, the abundance and distribution of virus-like particles (VLPs) associated with the SML and reef water of the coral Montipora capitata were determined using epifluorescence microscopy, while transmission electron microscopy was employed to determine the morphological diversity of VLPs. Sampling was conducted across the Coconut Island Marine Reserve (CIMR) reef system, Kane’ohe Bay, O’ahu, Hawai’i. Viral abundance was correlated with select environmental drivers and prokaryote abundance, while non-metric multidimensional scaling was used to determine the key environmental drivers of the viral community assemblage. The water column contained high concentrations of VLPs (5.98 × 107 ml-1) and prokaryotes (3.11 × 106 ml-1), consistent with the considerable anthropogenic impacts at this location. In comparison, the SML contained lower concentrations of VLPs (2.61 × 107 ml-1) and prokaryotes (2.08 × 106 ml-1); of note, the densities of viruses and prokaryotes in the SML were strongly coupled while those in the reef water were not. VLP density in the water column varied spatially across the reef, with the most sheltered site and the only one not situated on the reef crest having a greater VLP density than the other sites. Temporal variations in the density of microbes (i.e. viruses and prokaryotes) in the reef water were pronounced, while in the SML microbial densities remained constant. However, no specific environmental drivers of this variability could be identified. In contrast, temperature and water quality were correlated with shifts in the morphological diversity of VLPs across the reef. Small (< 50 nm) polyhedral/spherical VLPs were dominant, and were positively correlated to chlorophyll-a concentration when in the SML. In this same habitat, Fuselloviridae-like VLPs, filamentous VLPs and bead-shaped VLPs were positively correlated to temperature. In the reef water a different pattern was apparent: large (> 100 nm) Podoviridae-like VLPs and elongate Myoviridae-like VLPs, as well as lemon-shaped VLPs of both size classes showed positive associations with turbidity, while large filamentous VLPs, Geminiviridae-like VLPs and rod-shaped VLPs were positively associated with temperature. These results demonstrate that the viral community of Coconut Island’s reef is highly diverse, and subject to spatial and temporal change, especially in the water column. However, while the environmental drivers of viral diversity were partly elucidated, we are still a long way from understanding the drivers of viral abundance. More detailed study, both spatially and temporally, of the CIMR environment is required, as is comprehensive molecular analysis of the viral community of Kane’ohe Bay. Only then can we begin to understand the importance of viruses to the health and function of this, and other reef sites.</p>