scholarly journals A method for the collection of early-stage sea turtle embryos

2020 ◽  
Vol 42 ◽  
pp. 59-65
Author(s):  
A Gárriz ◽  
SA Williamson ◽  
RG Evans ◽  
RD Reina

Early-stage turtle embryos, immediately after oviposition, are very small (<5 mm diameter), hindering research on the initial period of embryonic development. For example, assessing whether turtle eggs had been fertilized and contained a viable embryo at oviposition, especially under field conditions, is complicated by the microscopic size of embryos that may have died at an early stage of development. Further, little is known about the molecular pathways that promote and regulate early developmental processes in turtles, such as pre-ovipositional embryonic arrest. To enable further investigation of the processes critical to early embryonic development in turtle species, a reliable method is required for extraction of early-stage embryos from the egg. Therefore, our aim was to develop a novel and reproducible method for extracting early-stage sea turtle embryos. Herein, we describe the technique for extracting Chelonia mydas embryos before and after white spot formation. Once the embryos were collected, the total RNA of 10 embryos was extracted to validate the method. The total RNA concentration was above 5 ng µl-1 and the RNA integrity number varied between 7.0 and 10.0, which is considered acceptable for further RNA-sequencing analyses. This extraction technique could be employed when investigating fertilization rates of turtle nests and for further investigation of the molecular biology of embryonic development in turtles. Furthermore, the technique should be adaptable to other turtle species or any oviparous species with similar eggs.

2019 ◽  
Vol 6 (1) ◽  
pp. 118
Author(s):  
Siti Zulaeha ◽  
Devit Purwoko ◽  
Imam Cartealy ◽  
Teuku Tajuddin ◽  
. Karyanti ◽  
...  

Comparison of Three RNA Extraction Kits for Transcriptome Analysis of Oil Palm (Elaeis guineensis Jacq.) ABSTRACTObtaining high-quality RNA is very important at an early stage of molecular biology research. To isolate RNA, high skill and caution are required in following laboratory procedures because RNA is easily degraded, especially samples from plant tissue culture. One of the parameters used to check the total RNA quality is RIN (RNA Integrity Number). The aim of this study was to obtain RNA extraction methods on oil palm leaves, callus and somatic embryos that were of good quality and high concentrations for transcriptomic analysis. RNA extraction was carried out using Plant RNA PureLink (Ambion), Genezol RNA Extraction (Geneaid) and RibospinTM Plant (Geneall) kit methods. The results showed that oil palm leaf, callus and somatic embryo RNA were successfully extracted using the RibospinTM (Geneall) kit. Based on the total RNA number of more than 4 μg and the RIN value of more than 7, the extracted RNA could be used in RNA sequencing for transcriptomic analysis. Keywords: callus, oil palm, RNA analysis, RNA quality, somatic embryo ABSTRAKMenghasilkan RNA berkualitas tinggi sangatlah penting pada tahap awal penelitian biologi molekuler. Untuk mengisolasi RNA diperlukan keterampilan dan kehati-hatian tinggi dalam mengikuti prosedur di laboratorium karena RNA lebih mudah terdegradasi, khususnya sampel hasil kultur jaringan tanaman. Salah satu parameter yang digunakan pada pengecekan kualitas RNA total adalah RIN (RNA Integrity Number). Penelitian bertujuan mendapatkan metode ekstraksi RNA pada daun, kalus dan embrio somatik kelapa sawit yang berkualitas baik dan memiliki konsentrasi tinggi untuk analisa transkriptomika.  Ekstraksi RNA dilakukan menggunakan metode kit Plant RNA PureLink (Ambion), Genezol RNA Extraction (Geneaid) dan RibospinTM Plant (Geneall). Hasil menunjukkan bahwa RNA daun, kalus dan embrio somatik kelapa sawit telah berhasil diekstraksi dengan menggunakan kit RibospinTM (Geneall). RNA hasil ekstraksi tersebut dapat digunakan untuk sekuensing RNA dengan tujuan analisis transkriptomika, dilihat dari jumlah total RNA yang lebih dari 4 μg dan nilai RIN lebih dari 7. Kata Kunci: analisis RNA, embrio somatic, kalus, kelapa sawit, kualitas RNA 


Author(s):  
Khairul Nizam Sehat ◽  
S. Vijay Kumar ◽  
Noor Hydayaty Md Yusuf

High-quality RNA is an important genetic study as it has minimal contaminants that can affect gene discovery including degraded RNAs, chemical, and biological residues. Hence, it is a prerequisite for genetic analysis using Next Generation Sequencing (NGS) for accurate and reliable data mining. Despite its importance, extracting high-quality RNA from different samples is often a challenge, as every tissue has a different biochemical composition, thus requiring different protocols. This paper reports protocols for the extraction of high-quality RNA from two type of pineapple tissues, which are thickly lignified hard tissue (tillers, inflorescence, flowers) and watery soft tissue (mature fruit, ripe fruit, and overripe fruit) via modified Kim and Hamada (2005) method. Total RNA was extracted in all six tissues, which showed two distinctive 25S and 18S band on agarose gel. The total RNA in this study was considered high-quality as the minimum concentration was 50 ng/μl, the absorbance ratio (A260:A280) was more than 1.8 and RNA integrity number (RIN) was greater than 7. The obtained results showed that the modified Kim and Hamada (2005) method was effective in extracting high-quality RNA from the challenging MD2 pineapple tissue, which is suitable for subsequent molecular analysis, including the highly sensitive NGS.


2020 ◽  
Author(s):  
David Terrington Booth ◽  
Alexander Archibald-Binge ◽  
Colin James Limpus

AbstractSea turtle embryos at high density nesting beaches experience relative high rates of early stage embryo death. One hypothesis to explain this high dead rate is that there is an increased probability that newly constructed nests are located close to maturing clutches whose metabolising embryos cause low oxygen levels, high carbon dioxide levels, and high temperatures. Although these altered environmental conditions are well tolerated by mature embryos, early stage embryos may not be as tolerant leading to an increase in their mortality. To test this hypothesis, we incubated newly laid sea turtle eggs for a week over a range of temperatures in different combinations of oxygen and carbon dioxide concentrations and assessed embryo development and death rates. We found that gas mixtures of decreased oxygen and increased carbon dioxide, similar to those found in natural sea turtle nest containing mature embryos, slowed embryonic development but did not influence embryo mortality of early stage embryos. In contrast, high incubation temperature not only decreased embryo development rate, but prolonged incubation at 34°C was fatal.


2021 ◽  
Author(s):  
Colin I. Elliott ◽  
Theresa E. Stotesbury ◽  
Aaron B.A. Shafer

AbstractDetermining the age, or time since deposition (TSD), of bloodstains would provide forensic scientists with critical information regarding the timeline of the events of bloodshed during a crime. The physicochemical changes that occur to major biomolecules as a bloodstain dries can be used to estimate the TSD of bloodstains. For example, high-resolution automated gel electrophoresis can be used to quantify the timewise degradation of DNA present in bloodstains. Our study aims to analyze and quantify the timewise degradation trends found in total RNA from bloodstains, expanding the scope of the TSD research which has previously explored DNA and targeted mRNA molecules. Fifty bloodstains were stored in plastic microcentrifuge tubes at room temperature and tested over 10 different timepoints spanning one week. A total of eight RNA metrics were visually assessed and quantified using linear regression. RNA Integrity Number equivalent (RINe), total RNA concentration, and 28S/18S rRNA peak area ratios were retained for further analyses based on their relationship with time and limited correlations. RINe and total RNA concentration both exhibited negative trends over time, highlighting a decrease in quality and quantity. RINe was the RNA metric that demonstrated the greatest association with time (R2 = 0.696). Generalized linear mixed-effects models including donor (biological replicate) as a random effect increased the fit for all RNA metrics to varying degrees, but no significant differences were found between biological replicates for the RINe metric. Our results illustrated the presence of a significant decrease in the retained RNA metrics after 24 hours, suggesting that this method could be used to reliably differentiate day-old bloodstains from older bloodstains. Future work should focus on recreating this study in different environmental conditions, including testing on a variety of substrates.


1971 ◽  
Vol 44 (1) ◽  
pp. 271-293 ◽  
Author(s):  
D. C. Prevorsek ◽  
W. James Lyons

Abstract The performance characteristics of simple filaments in fatigue in cyclic longitudinal tension are reviewed and discussed in terms of a theory which assumes that the fracture is a result of the formation of an unstable crack. It is shown that the derived relationships are in qualitative agreement with observed effects of temperature, frequency, stroke, etc. In quantitative studies however, and especially with those intended to extract values of unknown parameters, it must be observed that the derived expressions apply only for the conditions where the effects of structural reorganization in front of the propagating crack are negligible in comparison with the effects associated in the formation of new crack surfaces. Thus, the theory is applicable primarily to highly oriented fibers which are ruptured at temperatures below Tg. In the analysis of the results of the fatigue experiments, it is also necessary to take into account the structural changes which take place during the initial period of loading (mechanical conditioning). In this period the fibers change considerably in their properties (modulus, elongation at break, etc.) which in turn affects the fatiguing conditions. In the interpretation of data obtained in fatiguing at constant stress or strain amplitude, it must be observed that the theory also indicates that the severity of fatiguing conditions should be expressed in terms of strain-energy amplitude instead of the commonly used stress- or strain-amplitude arguments. This analysis is based on the appearance of the term σ2/E=σε in the expressions for lifetime. It is conceivable that our experimental data discussed in Experimental (fourth subsection) would not show the large difference between fatiguing at constant stroke and constant force-amplitude, if the results of both experiments were plotted as a function of σ∈. The most important goal of our study was to establish a method for predicting the potential endurance of fibers from their molecular structure. The derived equations include the three primary parameters which are affected by the molecular structure of the polymers: fracture surface energy, modulus, and activation energy associated with the processes involved in crack growth. The physical significance of these factors is discussed and methods to estimate their numerical values from known molecular parameters are reviewed. In correlating or predicting the fatigue behavior from molecular structure of the polymer, it must be remembered that the derived expressions hold for a perfectly oriented, flawless ensemble of molecules. The studies of fiber morphology on the other hand, show that the fibers consist of at least two phases differing primarily in the degree of order. Since the studies of mechanical coupling between phases indicate a poor load transfer between phases it is obvious that the morphological characteristics (e.g., chain folding) play a very important role in the overall mechanical behavior of the fibers and, therefore, must be considered. The studies of the effects of morphology on mechanical properties of fibers are still in an early stage of development. Further work is required to elucidate the fiber morphology and especially the structure of the phase boundary (crystal surfaces, concentration of tie-molecules, etc.). Developments are also necessary in a theory which would adequately describe the mechanical responses of such complex systems. If one considers that the strength of present “high tenacity” fibers is about 5–10 times lower than calculated values, assuming a flawless structure, then it is expected that functional modifications of fiber morphology should lead to significant increases in their strength, endurance, and modulus.


Author(s):  
Menghan TAO ◽  
Ning XIAO ◽  
Xingfu ZHAO ◽  
Wenbin LIU

New energy vehicles(NEV) as a new thing for sustainable development, in China, on the one hand has faced the rapid expansion of the market; the other hand, for the new NEV users, the current NEVs cannot keep up with the degree of innovation. This paper demonstrates the reasons for the existence of this systematic challenge, and puts forward the method of UX research which is different from the traditional petrol vehicles research in the early stage of development, which studies from the user's essence level, to form the innovative product programs which meet the needs of users and being real attractive.


Author(s):  
Renuka M. Tenahalli

Shweta Pradara (Leucorrhoea) is the disease which is characterized by vaginal white discharge. Vaginal white discharge this symptom is present in both physiological and pathological condition, when it becomes pathological it disturbs routine life style of the woman. Most of the women in the early stage will not express the symptoms because of hesitation and their busy schedule. If it is not treated it may leads to chronic diseases like PID (Garbhashaya Shotha etc.) Charaka mentioned Amalaki Choorna along with Madhu and Vata Twak Kashaya Yoni Pichu Dharana. This treatment is used in Shweta Pradara shown positive results, hence a study was under taken to assess its clinical efficacy. 30 diagnosed patients of Shweta Pradara were randomly selected, allocated in three groups. Group A and Group B received Amalaki Choorna with Madhu and Vata Twak Kashaya Yoni Pichu Dharana respectively and Group C received Amalaki Choorna with Madhu followed by Vata Twak Kashaya Yoni Pichu Dharana for 15 days. The patients were assessed for the severity of the symptoms subjectively and objectively before and after the treatment and at the end of the follow up. Data from each group were statistically analyzed and were compared. No side effects were noted and it may be considered as an effective alternative medicine in Shweta Pradara (leucorrhea). Amalaki is rich in natural source of vitamin C and contains phosphorus, iron and calcium. Honey contains carbohydrate, vitamin C, phosphorus iron and calcium. All together these help to increase Hb% and immunity. Vata Twak Kashaya contains tannin which helps to maintain normal pH of the vagina.


2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Danielle M. Nash ◽  
Zohra Bhimani ◽  
Jennifer Rayner ◽  
Merrick Zwarenstein

Abstract Background Learning health systems have been gaining traction over the past decade. The purpose of this study was to understand the spread of learning health systems in primary care, including where they have been implemented, how they are operating, and potential challenges and solutions. Methods We completed a scoping review by systematically searching OVID Medline®, Embase®, IEEE Xplore®, and reviewing specific journals from 2007 to 2020. We also completed a Google search to identify gray literature. Results We reviewed 1924 articles through our database search and 51 articles from other sources, from which we identified 21 unique learning health systems based on 62 data sources. Only one of these learning health systems was implemented exclusively in a primary care setting, where all others were integrated health systems or networks that also included other care settings. Eighteen of the 21 were in the United States. Examples of how these learning health systems were being used included real-time clinical surveillance, quality improvement initiatives, pragmatic trials at the point of care, and decision support. Many challenges and potential solutions were identified regarding data, sustainability, promoting a learning culture, prioritization processes, involvement of community, and balancing quality improvement versus research. Conclusions We identified 21 learning health systems, which all appear at an early stage of development, and only one was primary care only. We summarized and provided examples of integrated health systems and data networks that can be considered early models in the growing global movement to advance learning health systems in primary care.


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