Directional mapping of DNA nicking in ejaculated and cauda epididymidal spermatozoa of the short-beaked echidna (Tachyglossus aculeatus: Monotremata)

2009 ◽  
Vol 21 (8) ◽  
pp. 1008 ◽  
Author(s):  
S. D. Johnston ◽  
C. López-Fernández ◽  
A. Gosálbez ◽  
W. V. Holt ◽  
J. Gosálvez
Keyword(s):  
Sperm Nucleus ◽  
Sperm Head ◽  
Dna Conformation ◽  
Dna Breaks ◽  
Highly Sensitive ◽  
Dna Nicking ◽  
Klenow Polymerase ◽  
The Individual ◽  
Chromatin Rearrangement

Prototherian spermatozoa are unique amongst the Mammalia in terms of their filiform morphology, tandem arrangement of chromosomes and formation of sperm bundles. In the present study, we provide observations of echidna spermatozoa and note that the superstructure of the bundle is engineered around the shape of the individual sperm head and that this in turn may be a consequence of the unusual circumferential and helicoidal condensation of the DNA during spermiogenesis. Frozen–thawed ejaculated echidna spermatozoa were incubated and examined for the presence of non-typical DNA conformation by means of in situ labelling of DNA breaks using Klenow polymerase and via alkaline single-cell comet assays for detection of fragmented DNA. Both techniques successfully revealed the presence of what appeared to be directional DNA nicking, co-localised with the presence of highly sensitive alkali sites along the length of the sperm nucleus. It was not possible to define whether these alternative DNA configurations were associated with a failure of the sperm nucleus to condense appropriately during spermiogenesis or were evidence of DNA fragmentation following post-thaw incubation or a sequential structural chromatin rearrangement necessary for fertilisation.

scholarly journals Spatial expression pattern of serine proteases in the blood fluke Schistosoma mansoni determined by fluorescence RNA in situ hybridization

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Lenka Ulrychová ◽  
Pavel Ostašov ◽  
Marta Chanová ◽  
Michael Mareš ◽  
Martin Horn ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Schistosoma Mansoni ◽  
Rna Sequencing ◽  
Serine Proteases ◽  
Expression Patterns ◽  
High Sensitivity ◽  
Host Parasite Interactions ◽  
Highly Sensitive ◽  
Host Parasite

Abstract Background The blood flukes of genus Schistosoma are the causative agent of schistosomiasis, a parasitic disease that infects more than 200 million people worldwide. Proteases of schistosomes are involved in critical steps of host–parasite interactions and are promising therapeutic targets. We recently identified and characterized a group of S1 family Schistosoma mansoni serine proteases, including SmSP1 to SmSP5. Expression levels of some SmSPs in S. mansoni are low, and by standard genome sequencing technologies they are marginally detectable at the method threshold levels. Here, we report their spatial gene expression patterns in adult S. mansoni by the high-sensitivity localization assay. Methodology Highly sensitive fluorescence in situ RNA hybridization (FISH) was modified and used for the localization of mRNAs encoding individual SmSP proteases (including low-expressed SmSPs) in tissues of adult worms. High sensitivity was obtained due to specifically prepared tissue and probes in combination with the employment of a signal amplification approach. The assay method was validated by detecting the expression patterns of a set of relevant reference genes including SmCB1, SmPOP, SmTSP-2, and Sm29 with localization formerly determined by other techniques. Results FISH analysis revealed interesting expression patterns of SmSPs distributed in multiple tissues of S. mansoni adults. The expression patterns of individual SmSPs were distinct but in part overlapping and were consistent with existing transcriptome sequencing data. The exception were genes with significantly low expression, which were also localized in tissues where they had not previously been detected by RNA sequencing methods. In general, SmSPs were found in various tissues including reproductive organs, parenchymal cells, esophagus, and the tegumental surface. Conclusions The FISH-based assay provided spatial information about the expression of five SmSPs in adult S. mansoni females and males. This highly sensitive method allowed visualization of low-abundantly expressed genes that are below the detection limits of standard in situ hybridization or by RNA sequencing. Thus, this technical approach turned out to be suitable for sensitive localization studies and may also be applicable for other trematodes. The results suggest that SmSPs may play roles in diverse processes of the parasite. Certain SmSPs expressed at the surface may be involved in host–parasite interactions. Graphic abstract

scholarly journals Selected In Situ Hybridization Methods: Principles and Application

Molecules ◽  
2021 ◽  
Vol 26 (13) ◽  
pp. 3874
Author(s):  
Dominika Veselinyová ◽  
Jana Mašlanková ◽  
Katarina Kalinová ◽  
Helena Mičková ◽  
Mária Mareková ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Imaging Techniques ◽  
Cell Communication ◽  
Probe Design ◽  
Rapid Progress ◽  
In Situ Techniques ◽  
Different Types ◽  
Laboratory Procedures ◽  
The Individual

We are experiencing rapid progress in all types of imaging techniques used in the detection of various numbers and types of mutation. In situ hybridization (ISH) is the primary technique for the discovery of mutation agents, which are presented in a variety of cells. The ability of DNA to complementary bind is one of the main principles in every method used in ISH. From the first use of in situ techniques, scientists paid attention to the improvement of the probe design and detection, to enhance the fluorescent signal intensity and inhibition of cross-hybrid presence. This article discusses the individual types and modifications, and is focused on explaining the principles and limitations of ISH division on different types of probes. The article describes a design of probes for individual types of in situ hybridization (ISH), as well as the gradual combination of several laboratory procedures to achieve the highest possible sensitivity and to prevent undesirable events accompanying hybridization. The article also informs about applications of the methodology, in practice and in research, to detect cell to cell communication and principles of gene silencing, process of oncogenesis, and many other unknown processes taking place in organisms at the DNA/RNA level.

The Effect of Homoeologous Meiotic Pairing in Tetraploid Hordeum bulbosum L. × H. vulgare L. Hybrids on Alien Introgressions in Offspring

2016 ◽  
Vol 150 (2) ◽  
pp. 139-149 ◽  
Author(s):  
Margret Scholz ◽  
Galina Pendinen
Keyword(s):  
Interspecific Hybrids ◽  
In Situ Hybridisation ◽  
Basic Material ◽  
Meiotic Pairing ◽  
Hordeum Bulbosum ◽  
Homoeologous Pairing ◽  
Introgression Breeding ◽  
The Individual ◽  
Pairing Behaviour

The pairing behaviour of the individual chromosome arms of Hordeum vulgare (Hv) with their homoeologous arms of H. bulbosum (Hb) at metaphase I of meiosis in tetraploid Hb × Hv hybrids and the frequencies of recombined Hv chromosome arms in selfed offspring were studied on differentially visualized chromosomes after fluorescent in situ hybridisation. The frequencies of paired Hv-Hb arms in the F2 and F3 hybrids were correlated with the frequencies of recombined Hv chromosomes in progenies. Self-generation of hybrids, the number of Hv and Hb chromosomes, and the number of recombined Hv chromosomes of the hybrids strongly influenced the Hv-Hb pairing frequency in meiosis. Within the offspring of F2 and F3 hybrids both Hv plants and hybrids were detected. In contrast, all progenies of the F4 hybrid were hybrids which exhibited centromere misdivisions. The highest frequencies of homoeologous pairing in hybrids and most recombinants were obtained for the barley chromosome 1HL. Recombinants for 4HL, 5HS, 6HS, and 7HS were rarely found. Meiotic pairing and recombinants involving chromosome 1HS were never observed. The results of this study demonstrate that fertile tetraploid interspecific hybrids with a high intergenomic pairing at meiosis are valuable basic material for introgression breeding in barley.

A highly sensitive chlorine gas sensor and enhanced thermal DC electrical conductivity from polypyrrole/silicon carbide nanocomposites

RSC Advances ◽  
2016 ◽  
Vol 6 (87) ◽  
pp. 84200-84208 ◽  
Author(s):  
Adil Sultan ◽  
Sharique Ahmad ◽  
Faiz Mohammad
Keyword(s):  
Electrical Conductivity ◽  
Silicon Carbide ◽  
Gas Sensor ◽  
Dodecylbenzenesulfonic Acid ◽  
Chemical Polymerization ◽  
Dc Electrical Conductivity ◽  
Highly Sensitive ◽  
Chlorine Gas

We report the synthesis of polypyrrole (PPy) and polypyrrole/silicon carbide nanocomposites (PPy/SiC) and PPy/SiC/dodecylbenzenesulfonic acid (DBSA) by in situ chemical polymerization and their application as sensors for the detection of highly toxic chlorine gas.

scholarly journals Estimating long-term groundwater storage and its controlling factors in Alberta, Canada

2018 ◽  
Vol 22 (12) ◽  
pp. 6241-6255 ◽  
Author(s):  
Soumendra N. Bhanja ◽  
Xiaokun Zhang ◽  
Junye Wang
Keyword(s):  
Groundwater Recharge ◽  
Environmental Sustainability ◽  
Land Surface ◽  
Water Withdrawal ◽  
Groundwater Storage ◽  
Budget Analysis ◽  
Terrestrial Water ◽  
The Individual ◽  
Gravity Recovery

Abstract. Groundwater is one of the most important natural resources for economic development and environmental sustainability. In this study, we estimated groundwater storage in 11 major river basins across Alberta, Canada, using a combination of remote sensing (Gravity Recovery and Climate Experiment, GRACE), in situ surface water data, and land surface modeling estimates (GWSAsat). We applied separate calculations for unconfined and confined aquifers, for the first time, to represent their hydrogeological differences. Storage coefficients for the individual wells were incorporated to compute the monthly in situ groundwater storage (GWSAobs). The GWSAsat values from the two satellite-based products were compared with GWSAobs estimates. The estimates of GWSAsat were in good agreement with the GWSAobs in terms of pattern and magnitude (e.g., RMSE ranged from 2 to 14 cm). While comparing GWSAsat with GWSAobs, most of the statistical analyses provide mixed responses; however the Hodrick–Prescott trend analysis clearly showed a better performance of the GRACE-mascon estimate. The results showed trends of GWSAobs depletion in 5 of the 11 basins. Our results indicate that precipitation played an important role in influencing the GWSAobs variation in 4 of the 11 basins studied. A combination of rainfall and snowmelt positively influences the GWSAobs in six basins. Water budget analysis showed an availability of comparatively lower terrestrial water in 9 of the 11 basins in the study period. Historical groundwater recharge estimates indicate a reduction of groundwater recharge in eight basins during 1960–2009. The output of this study could be used to develop sustainable water withdrawal strategies in Alberta, Canada.

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