Live-dead Analysis Research Articles

Intrinsic insights to antimicrobial effects of Nitrofurantoin to multi drug resistant Salmonella enterica serovar Typhimurium ms202

Emerging multidrug resistant (MDR) serovar of Salmonella has raised the concern of their impactful effect on pathogenic infection and mortality in human lead by the enteric diseases. In order to combat the battle against these MDR Salmonella pathogen, new drug molecules need to be evaluated for their potent antibacterial application. This study evaluates the mechanistic antimicrobial effect of nitrofurantoin against a MDR strain of Salmonella named S. enterica Typhimurium ms202. The antimicrobial effect of nitrofurantoin was studied through experimental and computational approach using standard microbiological and molecular techniques like growth curve analysis, live-dead analysis, oxidative stress evaluation using high throughput techniques like flow cytometry and fluorescent microscopy. The result showed a potent dose dependent antibacterial effect of nitrofurantoin against S. enterica Typhimurium ms202 with a MIC value of 64 µg/ml. Moreover, the mechanistic excavation of the phenomenon described the mechanism as an effect of molecular interaction of nitrofurantoin molecule with membrane receptor proteins OmpC of S. enterica Typhimurium ms202 leading to internalization of the nitrofurantoin heading towards the occurrence of cellular physiological disturbances through oxidative stress impeded by nitrofurantoin-Sod1 C protein interaction. The results indicated towards a synergistic effect of membrane damage, oxidative stress and genotoxicity for the antibacterial effect of nitrofurantoin against S. enterica Typhimurium ms202. The study described the potent dose-dependent application of nitrofurantoin molecule against MDR strains of Salmonella and guided towards their use in further discovered MDR strains.

Developing Testable Hypotheses of Anthropogenic Stress: Some Approaches That Work

Conservation Paleobiology (CPB) has many aims, but ultimately depends upon our uncovering, for a target region, the history of environmental pressures and history of biological change, particularly biological change that signifies a response to anthropogenic stress: this is how we detect and correctly attribute deterioration, how we design and evaluate recovery, and how we ultimately assess resilience and sustainability. The focus of concern might be a single taxon of economic or other particular value, or larger-scale changes in biomass, taxonomic or phylogenetic diversity, or trophic complexity. One of the biggest challenges for natural scientists can be building the requisite history of cultural stressors – i.e., uncovering the diverse economic, industrial, social, and regulatory activities that might have affected the system. Such information is commonly not accessible via the Web of Science; it can be extremely important but qualitative or can be quantitative but highly variable in units or methods of measurement; and, with the exception of data on commercial harvesting (e.g., fishing, logging) and human population size, useful time-series are scarce. The CPB scientist thus typically needs to compile their own, original history of human activities having potential to affect natural systems, either to evaluate the (paleo)biological data that they already have on hand (from biomonitoring, live-dead analysis, sedimentary cores) or to frame a new campaign of data collection. Here, I describe approaches to finding and merging cultural data that have worked both for research and for class projects, using two coastal marine examples: (1) testing the effects of historical over-fishing (meta-analyses from the early 2000s), and (2) the unexpected role of land-use in the collapse of the open-shelf benthic ecosystem of southern California.

Death Assemblages Record Significant Range Contraction in a Molluscan Species of Concern from the Eastern Gulf Of Mexico

Marine habitats are in decline due to increasing anthropogenic pressures, but baseline data on species distributions needed to manage and conserve populations are lacking. Incorporating death assemblages into species assessments can create a more accurate understanding of pre-anthropogenic communities than survey records alone. In this study, we conducted a live-dead analysis on mollusks from a new 2008-2018 dredge survey in the eastern Gulf of Mexico. We selected the predatory banded tulip snail, Cinctura hunteria, as a test case for assessment because this species is one of several designated by the Florida Fish and Wildlife as a species of concern. Using spatial count data for shells in our samples, we estimated density values for each taxonomic grade over the sampled area using IDW spatial interpolation. These maps reveal large areas of occupation across the west Florida shelf for two taxonomic grades of dead shells but loss of offshore occurrence for live records. One explanation for the lack of occurrences in offshore habitats is that, unlike dead shell records, there is no time averaging accumulation of live shells. Time averaging increases detectability of species in habitats where they are rare. However, independent fisheries data from live-only animal surveys not only mirror our live-dead results but suggest that habitat loss in our live-dead comparisons was rapid and occurred in the late 1980s or early 1990s. Thus, live-dead comparisons reveal both natural baselines as well as anthropogenic changes in distribution without being significantly distorted by time-averaging biases. Including live-dead data can greatly improve species assessments when long-term survey records are unavailable and provide a key tool in combatting biodiversity loss across marine ecosystems.

Natural aging of expanded shale, clay, and slate (ESCS) amendment with heavy metals in stormwater increases its antibacterial properties: Implications on biofilter design

Aging is often expected to decrease the pathogen removal capacity of media because of exhaustion of attachment sites by adsorption of co-contaminants and dissolved organics. In contrast, the adsorption of metals naturally present in stormwater during aging could have a positive impact on pathogen removal. To examine the effect of adsorbed metals on pathogen removal, biofilter media amended with expanded clay, shale, and slate (ESCS) aggregates, a lightweight aggregate, were exposed to metals by intermittently injecting natural stormwater spiked with Cu, Pb, and Zn, and the capacity of aged and unaged media to remove Escherichia coli (E. coli), a pathogen indicator, were compared. Metal adsorption on ESCS media decreased their net negative surface charge and altered the surface properties as confirmed by zeta potential measurement and Fourier-Transform Infrared Spectroscopy (FTIR) analysis. These changes increased the E. coli adsorption capacity of aged media compared with unaged media and decreased overall remobilization of attached E. coli during intermittent infiltration of stormwater. A live-dead analysis confirmed that the adsorbed metals inactivated attached E. coli, thereby replenishing the adsorption capacity. Overall, the results confirmed that natural aging of biofilter media with adsorbed metals could indeed have a net positive effect on E. coli removal in biofilters and therefore should be included in the conceptual model predicting long-term removal of pathogens from stormwater containing mixed pollutants.

Oxidative stress induced antimicrobial efficacy of chitosan and silver nanoparticles coated Gutta-percha for endodontic applications

Root canal treatment is the most effective treatment for irreversible pulpal damage. However, it suffers the risk of failure due to micro-leakage at the Gutta-percha-sealer-tooth interface. Concerning the issue, it is important to enhance the antimicrobial properties of Gutta-percha. The current study describes a novel coating of the Gutta-percha with chitosan and silver nanoparticles to increase their antimicrobial efficacy. They were coated with chitosan and silver nanoparticles with concentrations of 1%, 2% using chemical methodology. Coated Gutta-percha were evaluated for their antimicrobial efficacy against E. faecalis, which is frequently isolated microorganism in failed endodontic cases using standard microbiological methods like growth curve analysis. Detailed analysis of the antimicrobial activity was performed by live-dead analysis with the help of flow cytometry and fluorescent microscopy. Although, the experimental analysis showed concentration-dependent antibacterial activity by both chitosan and silver NP coated Gutta-percha, the silver NP coating exhibited higher antibacterial activity compared to the Gutta percha coated with chitosan nanoparticles. Mechanistic evaluation unveiled the cause of antibacterial activates due to action of induced oxidative stress and membrane damage to the bacteria by coated nanoparticles. The information will be useful for the endodontist to have an alternative filling material with higher antibacterial potency for higher success rate of root canal therapies in clinical procedures.

Increasing the salience of marine live–dead data in the Anthropocene

AbstractAs the climate changes and ecosystems shift toward novel combinations of species, the methods and metrics of conservation science are becoming less species-centric. To meet this growing need, marine conservation paleobiologists stand to benefit from the addition of new, taxon-free benthic indices to the live–dead analysis tool kit. These indices, which were developed to provide actionable, policy-specific data, can be applied to the readily preservable component of benthic communities (e.g., mollusks) to assess the ecological quality status of the entire community. Because these indices are taxon-free, they remain applicable even as the climate changes and novel communities develop—making them a potentially valuable complement to traditionally applied approaches for live–dead analysis, which tend to focus on maintaining specific combinations of species under relatively stable environmental conditions. Integrating geohistorical data with these established indices has potential to increase the salience of the live–dead approach in the eyes of resource managers and other stakeholders.

Mimicking corneal stroma using keratocyte-loaded photopolymerizable methacrylated gelatin hydrogels.

Cell-laden methacrylated gelatin (GelMA) hydrogels with high (approximately 90%) transparency were prepared to mimic the natural form and function of corneal stroma. They were synthesized from GelMA with a methacrylation degree of 70% as determined by nuclear magnetic resonance. Hydrogels were strong enough to withstand handling. Stability studies showed that 87% of the GelMA hydrogels remained after 21days in phosphate buffered saline (PBS). Cell viability in the first 2days was over 90% for the human keratocytes loaded in the gels as determined with the live-dead analysis. Cells in the hydrogel elongated and connected to each other as observed by confocal laser scanning microscopy (CLSM) images and scanning electron microscope analysis after 3weeks in the culture medium and cells were seen to be distributed throughout the hydrogel bulk. Cells were found to synthesize collagen Types I and V, decorin, and biglycan (representative collagens and proteoglycans of human corneal stroma, respectively) showing that keratocytes maintained their functions and preserved their phenotypes in the hydrogels. Transparency of cell-loaded and cell-free hydrogels after 21days was found to be over 90% at all time points in the visible light range and was comparable to the transparency of the native cornea. The corneal stroma equivalent produced in this study that has cells entrapped in it leads to a product with homogenous distribution of cells. It was transparent at the very beginning and is expected to allow better vision than nontransparent substrates. It, therefore, has a significant potential to be used as an alternative to the current products used to treat corneal blindness.

Unrecognized loss of seagrass communities based on molluscan death assemblages: historic baseline shift in tropical Gulf of Aqaba, Red Sea

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 589:73-83 (2018) - DOI: https://doi.org/10.3354/meps12492 Unrecognized loss of seagrass communities based on molluscan death assemblages: historic baseline shift in tropical Gulf of Aqaba, Red Sea Ehud Gilad1,2, Susan M. Kidwell3, Yehuda Benayahu2, Yael Edelman-Furstenberg1,* 1Geological Survey of Israel, Jerusalem 95501, Israel 2Department of Zoology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel 3Department of Geophysical Sciences, University of Chicago, Chicago, IL 60637, USA *Corresponding author: yael@gsi.gov.il ABSTRACT: Anthropogenic stresses on the naturally oligotrophic Gulf of Aqaba over the last 50 yr are suspected to have had a strong impact on biota, but the status of benthic fauna on the extensive soft-sediment seafloors is unknown. This study represents the first research on possible anthropogenic impacts on benthic marine fauna other than corals in the Gulf of Aqaba, a region of exceptional biodiversity. Comparing the species and functional-group composition of the living bivalve community to dead-shell assemblages from the modern seabed (top 5 cm) at 15 and 30 m water depth, both proximal and distal to historic point sources of nutrients, reveals that the ecological baseline has shifted. Live-dead discordance is strongest in the area of former sewage outfall and fish farm operations where seagrass is now functionally absent, but molluscan trophic structure has changed significantly even at distal sites where seagrass is still moderately abundant. Chemosymbiont-bearing lucinid bivalves specialized to sandy seagrass dominate death assemblages but are scarce to absent in living assemblages, which are dominated instead by facultative deposit-feeding bivalves which prefer muddy sand or muddy seagrass, consistent with organic enrichment. Seagrass habitat has thus changed qualitatively even where it persists, and sites of seagrass loss have not recovered despite cessation of major pollution ≥10 yr ago. Although shells of dead-only species require geological age-dating to definitively establish human drivers, the documentation of significant habitat change and trophic restructuring makes a powerful case for adoption of paleontological analysis by environmental managers and conservation biologists. KEY WORDS: Live-dead analysis · Bivalves · Anthropogenic modification · Paleoecology · Gulf of Aqaba · Eutrophication Full text in pdf format Supplementary material PreviousNextCite this article as: Gilad E, Kidwell SM, Benayahu Y, Edelman-Furstenberg Y (2018) Unrecognized loss of seagrass communities based on molluscan death assemblages: historic baseline shift in tropical Gulf of Aqaba, Red Sea. Mar Ecol Prog Ser 589:73-83. https://doi.org/10.3354/meps12492 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 589. Online publication date: February 23, 2018 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2018 Inter-Research.

Live-dead analysis reveals long-term response of the estuarine bivalve community to water diversions along the Colorado River

Dam construction and water diversions along the Colorado River during the twentieth century have altered the downstream estuarine ecosystem. Although it is clear that the ecosystem has changed, a lack of pre-impact studies has made it challenging to determine the magnitude and direction of change. By using a paleontological approach, we can retrospectively estimate ecological conditions in the estuary from before dam construction, particularly for benthic organisms with preservable hard parts, such as the shells of bivalve mollusks. Here, for the first time, we quantify differences in evenness, richness, taxonomic similarity, and rank-order abundance between the bivalve community living in the estuary today and two potential geohistorical baselines, the accumulated dead shells on the tidal flat and an older accumulation – known to pre-date dam construction on the river – in nearby shell-rich beach ridges called cheniers.Analysis of more than 12,000 live and dead bivalves distributed among 18 species collected from the tidal flat and cheniers on Isla Montague – an island at the mouth of the Colorado River – indicates that the pre-dam bivalve community was dominated by Mulinia modesta and was characterized by low rarified species richness and evenness (indexed by Hurlbert’s Probability of Interspecific Encounter). The increase in these metrics in the live relative to the chenier assemblage reflects a shift in the estuary from brackish to full marine conditions after damming and water diversions reduced freshwater flow to the estuary during the twentieth century. The tidal flat death assemblage was more similar to the live assemblage, which suggests that it represents post-dam construction conditions due to a large input of recent shells, whereas the chenier assemblage is an appropriate pre-impact baseline. Taxonomic similarity and rank-order abundance, indexed by Jaccard-Chao and Spearman’s Rho metrics, respectively, were too conservative to detect these differences.These baseline data, most notably the documented changes between the live and chenier assemblages, can be used to assess the efficacy of restoration efforts in the estuary, such as the Minute 319 environmental pulse flows. If restoration efforts stimulate a response in the bivalve community, metric values for evenness and richness are expected to decrease and become more similar to those we report here for the pre-impact chenier assemblage.

The Chromosomal parDE2 Toxin-Antitoxin System of Mycobacterium tuberculosis H37Rv: Genetic and Functional Characterization.

Mycobacterium tuberculosis H37Rv escapes host-generated stresses by entering a dormant persistent state. Activation of toxin-antitoxin modules is one of the mechanisms known to trigger such a state with low metabolic activity. M. tuberculosis harbors a large number of TA systems mostly located within discernible genomic islands. We have investigated the parDE2 operon of M. tuberculosis H37Rv encoding MParE2 toxin and MParD2 antitoxin proteins. The parDE2 locus was transcriptionally active from growth phase till late stationary phase in M. tuberculosis. A functional promoter located upstream of parD2 GTG start-site was identified by 5′-RACE and lacZ reporter assay. The MParD2 protein transcriptionally regulated the PparDE2 promoter by interacting through Arg16 and Ser15 residues located in the N-terminus. In Escherichia coli, ectopic expression of MParE2 inhibited growth in early stages, with a drastic reduction in colony forming units. Live-dead analysis revealed that the reduction was not due to cell death alone but due to formation of viable but non-culturable cells (VBNCs) also. The toxic activity of the protein, identified in the C-terminal residues Glu98 and Arg102, was neutralized by the antitoxin MParD2, both in vivo and in vitro. MParE2 inhibited mycobacterial DNA gyrase and interacted with the GyrB subunit without affecting its ATPase activity. Introduction of parE2 gene in the heterologous M. smegmatis host prevented growth and colony formation by the transformed cells. An M. smegmatis strain containing the parDE2 operon also switched to a non-culturable phenotype in response to oxidative stress. Loss in colony-forming ability of a major part of the MParE2 expressing cells suggests its potential role in dormancy, a cellular strategy for adaptation to environmental stresses. Our study has laid the foundation for future investigations to explore the physiological significance of parDE2 operon in mycobacterial pathogenesis.

Biocompatibility of common polyimides with human endothelial cells for a cardiovascular microsensor.

The cardiovasculature is an emerging niche for polyimide microdevices, yet the biocompatibility of polyimides with human endothelial cells has not been reported in the literature. In this study, we have evaluated an experimental polyimide-based pressure sensor for biological safety to determine its suitability for intravascular operation by using an in vitro model of human endothelium, following ISO 10993-5 protocols for extract tests and direct contact tests. First, SV-HCEC cells were incubated with extracts derived from common microfabrication polyimides utilized in the transducer (PMDA-ODA, BPDA-PPD, and a proprietary thermoplastic adhesive), and then labeled with selective probes to evaluate the effect of the polyimides on mitochondria and cell viability. Flow cytometry analysis showed that incubation of SV-HCECs with polyimide extracts resulted in no significant change in mitochondrial membrane potential (detected by JC-1) or apoptotic (annexin V) and necrotic (propidium iodide) cell death, when compared to incubation with extracts of high-density polyethylene (HDPE) and untreated cells used as negative controls. Second, primary human endothelial cells were incubated in direct contact with the completed sensor and then labeled with selective probes for live-dead analysis (calcein-AM, ethidium homodimer-1). Endothelial cells showed no loss of viability when compared to negative controls. Combined, the studies show no significant change in early markers of stress or more strict markers of viability in endothelial cells treated with the polyimides tested. We conclude that these common microfabrication polyimides and the derived sensor are not cytotoxic to human endothelial cells, the primary cell type that cardiovascular sensors will contact in vivo.

In vitro evaluation of bioactive strontium-based ceramic with rabbit adipose-derived stem cells for bone tissue regeneration

The development of bone replacement materials is an important objective in the field of orthopaedic surgery. Due to the drawbacks of treating bone defects with autografts, synthetic bone graft materials have become optional. So in this work, a bone tissue engineering approach with radiopaque bioactive strontium incorporated calcium phosphate was proposed for the preliminary cytocompatibility studies for bone substitutes. Accumulating evidence indicates that strontium containing biomaterials promote enhanced bone repair and radiopacity for easy imaging. Hence, strontium calcium phosphate (SrCaPO4) and hydroxyapatite scaffolds have been investigated for its ability to support and sustain the growth of rabbit adipose-derived mesenchymal stem cells (RADMSCs) in vitro. They were characterized via Micro-CT for pore size distribution. Cells used were isolated from New Zealand White rabbit adipose tissue, characterized by FACS and via differentiation into the osteogenic lineage by alkaline phosphatase, Masson's trichome, Alizarin Red and von Kossa staining on day 28. Material-cell interaction was observed by SEM imaging of cell morphology on contact with material. Live-Dead analysis was done by confocal laser scanning microscopy and cell cluster analysis via μCT. The in vitro biodegradation, elution and nucleation of apatite formation of the material was evaluated using simulated body fluid and phosphate buffered saline in static regime up to 28 days at 37 °C. These results demonstrated that SrCaPO4 is a good candidate for bone tissue engineering applications and with osteogenically-induced RADMSCs, they may serve as potential implants for the repair of critical-sized bone defects.

Invasion of Laguncula pulchella (Gastropoda: Naticidae) and predator–prey interactions with bivalves on the Tona coast, Miyagi prefecture, northern Japan

On the Tona coast, Miyagi prefecture, northern Japan, interactions between the alien predator Laguncula pulchella and its bivalve prey were explored using annually collected quadrat samples over a 10 year period, from 2001 to 2010. A single L. pulchella individual was first recorded in 2002, and the density increased 12-fold from 2002 to 2004. In contrast, population densities of Ruditapes philippinarum and Macoma incongrua rapidly decreased during this interval. Based on frequency of predatory drill holes on the dead shells, more than 35 % of Ruditapes philippinarum and 20 % of Macoma incongrua died because of naticid predation after 2004, while Pillucina pisidium was less vulnerable to naticid predation. L. pulchella focused attacks on P. pisidium in 2004, when R. philippinarum and M. incongrua had became scarce due to naticid predation. This species-selective predation affected bivalve community structure, and caused disagreements in taxonomic composition and species’ rank-order abundance between the living bivalve community and the assemblage of dead shells. This approach (live–dead analysis), frequently used in paleoecological research, is a conservative tool to identify impacts of an alien predator on community structure. When sample size is sufficient, frequency of predatory drill holes in preferred prey species is likely to reflect predation intensity.

Multi parameter in vitro testing of ratjadone using flow cytometry.

Ratjadone, isolated from the myxobacterium Sorangium cellulosum, belongs to the family of so-called orphan ligands, which includes leptomycin, callystatin and other compounds. In previous screening tests, ratjadone revealed a growth inhibitory effect against bacteria, yeast and human cancer cells. Following these first results, ratjadone was tested on several human tumour cell lines (Jurkat, HepG2, U87-MG) and, as a control, on a non-tumour cell line (RLC18) for its mode of action. The cell analysis was carried out by flow cytometry. This comprised cell density measurements, live-dead analysis, cell-cycle analysis and detection of apoptosis. First experiments confirmed the growth inhibitory effect on any chosen tumour cell line. Following these results a dose effect relationship was monitored, confirming the high effectiveness of ratjadone against cell growth at nanomolar concentration. Cell cycle analysis has shown that ratjadone intervenes in the cell cycle by arresting the cells in G1-phase. Biological testing of additional ratjadone derivatives with changed configuration and stereochemistry, identified the pharmacophoric site of the molecule.

The Survivability of Mycoplasma meleagridis in Frozen-Thawed Turkey Semen

Semen from 15 turkeys was pooled and divided into two groups and diluted with glycerated freezing medium. One group contained naturally occurring levels of Mycoplasma meleagridis while the other was inoculated with a 24-hr culture of the organism. Both groups were frozen under identical conditions. Semen was evaluated for motility and live-dead analysis during various stages of cryopreservation. The number of viable mycoplasmas in both groups was determined prior to and at 1-, 2-, and 6-months frozen storage. The experiment was repeated twice.Semen motility and live-dead evaluation showed no apparent trends outside of the normal decline seen during various stages of cryopreservation. There was no significant decline in M. meleagridis levels in either treatment group in either trial when samples were tested at intervals up to 6 months. The naturally infected semen contained approximately 103 cfu/ml, while the inoculated semen had 105 cfu/ml. It was concluded that viable numbers of M. meleagridis do not substantially decline in turkey semen during cryopreservation and subsequent thawing. Consideration must be given to potential pathogens in turkey semen cryopreserved for long-term storage.