Lysis Of Eggs Research Articles

Effects of multi-frequency ultrasound on inactivating Chironomus kiiensis eggs: Evidence from a case study in drinking water treatment plant

Chironomid is a type of arthropod that lay eggs on objects in stagnant waters. Vertical side walls of sedimentation tanks in drink water treatment plant (DWTP) are an ideal place for it, which often leads to outbreaks of the larvae booming. Poor hygiene of drinking water and high risk of vector-borne disease are often related to maggots and adult Chironomus, which need to be effectively inhibited. Herein, a study was performed to investigate the effects of single-, dual- and triple-frequency ultrasonic radiation for the stress responses of Chironomus eggs on their viabilities, stress-related protein concentrations and antioxidative enzyme activities. Compared to the single- and dual- frequency treatments, the maximum acoustic pressure ranged from −30 pa to 12 Pa was achieved by the triple - frequency treatment. Meanwhile, the lowest integrity (25.63 %) and hatching rate (2.6 %) of Chironomus eggs were recorded after 4-min of triple-frequency treatment with 10-cm exposure distance and 450 W power. As the introduction of different frequency ultrasound, hyperoxide was generated from the effect of transient cavitation, which significant reduced activities on acetylcholinesterase (AchE), cytochrome P450, and glutathione S-transferases (GSTs) (p < 0.05), and were responsible for lysis of eggs' periplasm and shells. The work provided a commercial-scale case study to inhibit Chironomus biomass and prevent its infestation in DWTP.

Optimized DNA-based identification of Toxocara spp. eggs in soil and sand samples

BackgroundToxocara canis and Toxocara cati are globally distributed roundworms and causative agents of human toxocariasis, via ingestion of Toxocara eggs. Control of Toxocara infections is constrained by a lack of sensitive methods for screening of animal faeces and environmental samples potentially contaminated by Toxocara eggs. In this work, a pre-analytical method for efficient extraction of DNA from Toxocara eggs in environmental samples was set up using our previously validated T. canis- and T. cati-specific quantitative real-time polymerase chain reaction (qPCR). For this purpose, the influence of different methods for egg lysis, DNA extraction and purification for removal of PCR inhibitors were assessed on environmental samples.MethodsTo select the best egg disruption method, six protocols were compared on pure T. canis egg suspensions, including enzymatic lysis and thermal or mechanical disruption. Based on the selected best method, an analytical workflow was set up to compare two DNA extraction methods (FastDNA™ SPIN Kit for Soil versus DNeasy® PowerMax® Soil Kit) with an optional dilution and/or clean-up (Agencourt® AMPure®) step. This workflow was evaluated on 10-g soil and 10-g sand samples spiked with egg suspensions of T. canis (tenfold dilutions of 104 eggs in triplicate). The capacity of the different methods, used alone or in combination, to increase the ratio of positive tests was assessed. The resulting optimal workflow for processing spiked soil samples was then tested on environmental soil samples and compared with the conventional flotation-centrifugation and microscopic examination of Toxocara eggs.ResultsThe most effective DNA extraction method for Toxocara eggs in soil samples consisted in the combination of mechanical lysis of eggs using beads, followed by DNA extraction with the DNeasy® PowerMax® Soil Kit, and completed with an additional DNA clean-up step with AMPure® beads and a sample DNA dilution (1:10). This workflow exhibited a limit of detection of 4 and 46 T.canis eggs in 10-g sand and 10-g soil samples, respectively.ConclusionsThe pre-analytical flow process developed here combined with qPCR represents an improved, potentially automatable, and cost-effective method for the surveillance of Toxocara contamination in the environment.Graphical

Point of care colourimetric and lateral flow LAMP assay for the detection of Haemonchus contortus in ruminant faecal samples.

In this study, we present an optimised colourimetric and a lateral flow LAMP assay for the detection of Haemonchus contortus in small ruminant faecal samples. Using a previously published LAMP primer set, we made use of commercially available colourimetric LAMP and lateral flow kits and combined this into an optimised diagnostic assay which was then tested on field faecal samples from Eastern and South-Eastern Hungary as well as a pure H. contortus egg faecal sample from Košice, Slovakia. Both assays showed no conflicts in visual detection of the results. Additionally, we modified and tested several centrifuge-free DNA extraction methods and one bead-beating egg lysis DNA extraction method to develop a true point of care protocol, as the source of the starting DNA is the main rate-limiting step in farm-level molecular diagnosis. Out of the various methods trialed, promising results were obtained with the magnetic bead extraction method. Sample solutions from the Fill-FLOTAC® technique were also utilised, which demonstrated that it could be efficiently adapted for field-level egg concentration to extract DNA. This proof of concept study showed that isothermal amplification technologies with a colourimetric detection or when combined with a lateral flow assay could be an important step for a true point of care molecular diagnostic assay for H. contortus.

Egg Lysis Buffer Salts (ELB Salts; 10×)

Egg Lysis Buffer Salts (ELB Salts; 10×)

Egg Lysis Buffer (ELB-Sucrose)

Egg Lysis Buffer (ELB-Sucrose)

Egg Lysis Buffer–Chromatin Isolation Buffer (ELB–CIB)

Egg Lysis Buffer–Chromatin Isolation Buffer (ELB–CIB)

Development of a Biosensor-Based Rapid Urine Test for Detection of Urogenital Schistosomiasis.

Schistosomiasis affects up to 300 million people in regions of South America, Southeast Asia, the Middle East, Mediterranean Europe, and sub-Saharan Africa. Chronic consequences of schistosomiasis include anemia, physical and cognitive retardation in children, and organ failure. With predilection for the genitourinary tract, Schistosoma haematobium increases the risk of bladder cancer and HIV infection in women. Microscopic identification and enumeration of parasite eggs in urine (S. haematobium) or stool (Schistosoma mansoni and Schistosoma japonicum) has remained the standard for schistosomiasis diagnosis [1]. Limitations of microscopy include the need for skilled personnel in the field with microscopy equipment and its time-consuming nature. Particularly in infrastructure-limited regions, point-of-care (POC) molecular diagnostics hold the potential to transform the management of infectious diseases such as schistosomiasis that carry significant long-term morbidity if left undiagnosed. POC diagnosis could allow selective drug administration to individuals with confirmed infection rather than entire schools or communities, and an integrated diagnosis–single dose therapy approach could reduce costs of drug administration campaigns and minimize treatment-associated adverse effects. Electrochemical biosensors are well suited for molecular diagnostics because of their high sensitivity, low cost, ease of integration into POC devices, and portability of the reader instrumentation [2]. We have developed a strategy for rapid (one hour) molecular diagnosis of bacterial urinary tract infections using electrochemical biosensors [3,4]. Urinary cells are lysed and directly applied to an array of sensors functionalized with oligonucleotide probes targeting the 16S ribosomal RNA (rRNA) of common uropathogens [3,4]. Formation of the sequence-specific hybridization complex between the pathogen rRNA and the labeled capture and detector probe pairs is detected by an enzyme tag that mediates an amperometric signal output (Fig 1). Fig 1 Biosensor-based molecular detection of urinary pathogen. In this work, we demonstrate the use of our biosensor-based platform for detection of S. haematobium in urine. We developed capture and detector probes targeting S. haematobium rRNA and integrated them into our established molecular diagnostics strategy. After determining an efficient egg lysis strategy, we demonstrated direct electrochemical detection of S. haematobium eggs spiked in human urine.

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

PP1 control of M phase entry exerted through 14-3-3-regulated Cdc25 dephosphorylation

It has been known for over a decade that inhibition of protein phosphatase 1 (PP1) activity prevents entry into M phase, but the relevant substrate has not been identified. We report here that PP1 is required for dephosphorylation of the Cdc2-directed phosphatase Cdc25 at Ser287 (of Xenopus Cdc25; Ser216 of human Cdc25C), a site that suppresses Cdc25 during interphase. Moreover, PP1 recognizes Cdc25 directly by interacting with a PP1-binding motif in the Cdc25 N-terminus. We have also found that 14-3-3 binding to phospho-Ser287 protects Cdc25 from premature dephosphorylation. Upon entry into M phase, 14-3-3 removal from Cdc25 precedes Ser287 dephosphorylation, suggesting the existence of a phosphatase- independent pathway for 14-3-3 removal from Cdc25. We show here that this dissociation of 14-3-3 from Cdc25 requires the activity of the cyclin-dependent kinase Cdk2, providing a molecular explanation for the previously reported requirement for Cdk2 in promoting mitotic entry. Collectively, our data clarify several steps important for Cdc25 activation and provide new insight into the role of PP1 in Cdc2 activation and mitotic entry.

Zinc increases the longevity of unfertilized sea urchin eggs

We have found that Zn2+ prevented lysis of unfertilized sea urchin eggs, and the eggs retained the ability to form fertilization membranes and to divide. SDS polyacrylamide gel electrophoresis showed that proteolysis of several proteins accompanied egg lysis, but Zn2+ inhibited this proteolysis. Therefore, Zn2+ blocks protease activity directly or indirectly and thereby prolongs the longevity of unfertilized sea urchin eggs.

TAME stabilizes the cortex and mitotic apparatus of the sea urchin egg during isolation

The synthetic substrate p-tosyl- l-arginine methyl ester (TAME) has been included in buffered EGTA media used for the isolation of the mitotic apparatus from clam eggs and also for the isolation of the cortex from sea urchin eggs. In the course of an investigation of the role of actin-fascin and actin-myosin interactions in cytokinesis, the isolation of the sea urchin egg cortex was re-examined and the stability of the cortex to lysis in a buffered EGTA medium near neutrality found to depend directly on the presence of TAME. Lysis of eggs at metaphase in this medium yielded a mixture of cortices and mitotic apparatuses (MA); MA stability under these conditions also required the presence of TAME, although a reduced pH allowed MA isolation in its absence. The action of TAME in stabilizing the actin-based structure of the cortex and the microtubule-based structure of the MA is not duplicated by other proteolysis inhibitors and this compound will also induce actin polymerization and gelation in extracts of the soluble cytoplasmic proteins of the egg under conditions where these are normally inhibited.

Attack of sea urchin eggs by dogfish phagocytes: Model of phagocyte-mediated cellular cytotoxicity

To test whether lysosomal degranulation of phagocytes is associated with antibody-dependent cytotoxicity, eggs of Arbacia punctulata were used as targets for blood phagocytes of Mustelus canis. Eggs were coated with heat-aggregated dogfish IgM and exposed to phagocytes, and cytolysis of eggs was observed by Nomarski optics. Phagocytes adhered, degranulated, and raised fertilization membranes resembling those induced by sperm or ionophore A23187. Lysis was then observed as damage radiating from the point of phagocyte-egg contact. By 4 hr, coated eggs exposed to phagocytes released 8.9, 12.3, and 7.4% of total catalase (EC 1.11.1.6), beta-glucuronidase (EC 3.2.1.31), and superoxide dismutase (EC 1.15.1.1) into the medium. Cytotoxic enzyme release significantly exceeded that from uncoated eggs incubated with phagocytes or eggs alone (uncoated or coated). Because activated eggs release a neutral protease, it was considered possible that this enzyme might be responsible for autolysis of eggs. This possibility was excluded because (i) lysis of eggs was not inhibited by soybean trypsin inhibitor (SBTI) whereas the egg protease was sensitive to SBTI, and (ii) the major trypsin-like activity of phagocytes was not inhibited by SBTI. These experiments demonstrate that Ig-coated cells are first activated, and then killed, when exposed to degranulating phagocytes and suggest that enzymes from attacking phagocytes, and not target cells, are responsible for cell death.