1,721,025 research outputs found
Age-associated changes in transcriptional elongation and their effects on homeostasis
No full texthttp://dx.doi.org/10.13039/501100004189 Max Planck Societyhttp://dx.doi.org/10.13039/501100001659 Deutsche Forschungsgemeinschaf
Sperm cryopreservation and in vitro fertilization techniques for the African turquoise killifish Nothobranchius furzeri
Full text linkOver the last decade, the African turquoise killifish, Nothobranchius furzeri, has emerged as an important model system for the study of vertebrate biology and ageing. Propagation of laboratory inbred strains of Nothobranchius furzeri, such as GRZ, however, can pose challenges due to the short window of fertility, the efforts and space requirements involved in continuous strain maintenance, and the risks of further inbreeding. The current method for long term strain preservation relies on arrest of embryos in diapause. To create an alternative for long term maintenance, we developed a robust protocol to cryopreserve and revive sperm for in vitro fertilization (IVF). We tested a variety of extender and activator buffers for sperm IVF, as well as cryoprotectants to achieve practical long-term storage and fertilization conditions tailored to this species. Our protocol enabled sperm to be preserved in a cryogenic condition for months and to be revived with an average of 40% viability upon thawing. Thawed sperm were able to fertilize nearly the same number of eggs as natural fertilization, with an average of ~ 25% and peaks of ~ 55% fertilization. This technical advance will greatly facilitate the use of N. furzeri as a model organism
Micro-CT Analysis of Fat in the Killifish Nothobranchius furzeri
No full textAging is associated with an increase in body fat mass and a concomitant decrease in lean mass and bone density in mammals. Body adiposity can also be redistributed with age, resulting in abdominal fat accumulation and subcutaneous fat reduction. In addition, specific variation in fat distribution is considered to be a risk factor for a number of age-related metabolic disorders. Micro computed tomography (micro-CT) is a nondestructive high-resolution imaging method that uses planar X-ray images captured at various angles around a sample of interest to yield a three-dimensional array of radiodensity values, which can then be used to computationally extract the adipose volume in situ using its innate contrast properties. This method was successfully used to study adipose tissue dynamics in rodents and more recently in zebrafish. The naturally short-lived African turquoise killifish is an emerging model organism to study the biology of aging. Like mammals, killifish also have different fat deposits (visceral and subcutaneous), making them a suitable model to study age-related changes in fat mass and distribution. However, procedures allowing precise quantification of fat content and distribution are missing in this species. Here, we provide an optimized protocol to measure and quantify fat distribution in turquoise killifish by micro-CT scan analysis and show the applicability of the method in young and old animals of both sexes
Sperm Cryopreservation of the African Turquoise KillifishNothobranchius furzeri
No full textSperm cryopreservation is an essential method for the genetic preservation and long-Term storage of wild-Type and transgenic animal stocks. In addition, it allows for the synchronization of gamete availability and the transport and sharing of lines between different laboratories. Here, we describe a protocol developed in our laboratory for the extraction and cryopreservation of killifish (Nothobranchius furzeri) sperm
Nonlethal Blood Sampling from the Killifish Nothobranchius furzeri
Full text linkBlood withdrawal is a common procedure performed on laboratory animals to monitor key processes and indicators of fish health and physiology, such as hematopoiesis, hemostasis, and lipid and glucose metabolism. Moreover, the ability to extract blood with minimal invasiveness and without sacrificing animals enables repeated sampling, allowing both longitudinal studies of individual animals, as well as reducing the number of experimental animals needed in a study. The African turquoise killifish is an emerging animal model that is progressively being adopted worldwide for aging studies because of its naturally short life span. However, because of the small body size of this species, nonlethal blood collection is a challenging procedure. Here we present a detailed protocol enabling repeated blood sampling from the same individual fish. This method, if correctly executed, is minimally invasive and does not cause any lasting damage. The protocol has been tested on animals spanning from 6 to 24 wk of age and the amount of blood that could be extracted varied from 0.5 to 8 μL, greatly depending on specimen age, sex, and size. This volume is sufficient to perform analyses such as blood glucose measurement, blood cell counts, or histological stains on blood smears
In Vitro Fertilization of the African Turquoise KillifishNothobranchius furzeri
Full text linkThe ability to perform in vitro fertilization, together with sperm cryopreservation, greatly facilitates the long-term laboratory maintenance of wild-type and transgenic model organisms and helps prevent genetic drift. It is also useful in cases where reproduction may be compromised. In this protocol, we present a method for in vitro fertilization of the African Turquoise killifish Nothobranchius furzeri that is compatible with the use of fresh or cryopreserved sperm
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Uloga DAF-41 u životnom vijeku Caenorhabditis elegans
No full textTiku et al. (2017) published that loss of the protein NCL-1 (abnormal NuCLeoli 1) abolishes
the extended lifespan in major Caenorhabditis elegans longevity pathways, including the
germline-deficient glp-1 (abnormal Germ Line Proliferation 1) C. elegans mutant. Recent
unpublished work from the Antebi group identified a protein DAF-41 (Abnormal Dauer
Formation 41), an HSP-90 (heat shock protein 90) co-chaperone, as a potential downstream
regulator of glp-1ncl-1 lifespan. I investigated the role of daf-41 in glp-1ncl-1 lifespan, finding
that daf-41 downregulation extended the lifespan of glp-1ncl-1 worms. Moreover, I performed
RT-qPCR analysis of genes associated with the insulin signaling pathway (daf-16, lipl-4, sod3, dod-3), heat-shock response (hsf-1, hsp-70, hsp-16.2, hsp-4), and steroid pathway (daf-12,
fard-1, cdr-6, daf-36), quantified GFP-tagged HSF-1 and DAF-16 transcription factor activity
and measured thermotolerance and oxidative stress resistance in different mutant backgrounds.
Changes in gene expression, DAF-16 and HSF-1 activity, thermotolerance, and oxidative stress
resistance did not clearly explain the mechanism behind this lifespan extension.Tiku i suradnici (2017) primijetili su da delecija gena tumor-supresora NCL-1 (eng. Nucleoli
1) skraćuje produljeni životni vijek u više dugovječnih modela Caenorhabditis elegans, među
kojima i u dvostrukom mutantu glp-1ncl-1 (eng. Abnormal Germ Line Proliferation 1).
Prethodno neobjavljena istraživanja Antebija i suradnika nedavno su identificirala protein
DAF-41 (eng. Abnormal Dauer Formation 41), ko-šaperon proteina HSP-90 (eng. Heat Shock
Protein 90) kao potencijalnog nizvodnog regulatora životnog vijeka mutanta glp-1ncl-1.
Otkrila sam da utišavanje gena daf-41 produžuje životni vijek C. elegans mutanta glp-1ncl-1.
Kako bih otkrila potencijalni signalni put ovog fenomena, provela sam RT-qPCR analizu gena
inzulinskog signalnog puta (daf-16, lipl-4, sod-3, dod-3), odgovora na toplinski šok (hsf-1, hsp70, hsp-16.2, hsp-4) i steroidnog signalnog puta (daf-12, fard-1, cdr-6, daf-36). Zatim sam
kvantificirala aktivnost fluorescentno obilježenih transkripcijskih faktora HSF-1 i DAF-16 te
mjerila otpornost oblića na toplinski i oksidativni stres. Međutim, promjene u ekspresiji gena,
aktivnosti DAF-16 i HSF-1, otpornost na toplinski i oksidativni stres nisu dali jasan uvid u
mehanizam produženja životnog vijeka mutanata glp-1ncl-1 s utišanim genom daf-41
Uloga DAF-41 u životnom vijeku Caenorhabditis elegans
No full textTiku et al. (2017) published that loss of the protein NCL-1 (abnormal NuCLeoli 1) abolishes
the extended lifespan in major Caenorhabditis elegans longevity pathways, including the
germline-deficient glp-1 (abnormal Germ Line Proliferation 1) C. elegans mutant. Recent
unpublished work from the Antebi group identified a protein DAF-41 (Abnormal Dauer
Formation 41), an HSP-90 (heat shock protein 90) co-chaperone, as a potential downstream
regulator of glp-1ncl-1 lifespan. I investigated the role of daf-41 in glp-1ncl-1 lifespan, finding
that daf-41 downregulation extended the lifespan of glp-1ncl-1 worms. Moreover, I performed
RT-qPCR analysis of genes associated with the insulin signaling pathway (daf-16, lipl-4, sod3, dod-3), heat-shock response (hsf-1, hsp-70, hsp-16.2, hsp-4), and steroid pathway (daf-12,
fard-1, cdr-6, daf-36), quantified GFP-tagged HSF-1 and DAF-16 transcription factor activity
and measured thermotolerance and oxidative stress resistance in different mutant backgrounds.
Changes in gene expression, DAF-16 and HSF-1 activity, thermotolerance, and oxidative stress
resistance did not clearly explain the mechanism behind this lifespan extension.Tiku i suradnici (2017) primijetili su da delecija gena tumor-supresora NCL-1 (eng. Nucleoli
1) skraćuje produljeni životni vijek u više dugovječnih modela Caenorhabditis elegans, među
kojima i u dvostrukom mutantu glp-1ncl-1 (eng. Abnormal Germ Line Proliferation 1).
Prethodno neobjavljena istraživanja Antebija i suradnika nedavno su identificirala protein
DAF-41 (eng. Abnormal Dauer Formation 41), ko-šaperon proteina HSP-90 (eng. Heat Shock
Protein 90) kao potencijalnog nizvodnog regulatora životnog vijeka mutanta glp-1ncl-1.
Otkrila sam da utišavanje gena daf-41 produžuje životni vijek C. elegans mutanta glp-1ncl-1.
Kako bih otkrila potencijalni signalni put ovog fenomena, provela sam RT-qPCR analizu gena
inzulinskog signalnog puta (daf-16, lipl-4, sod-3, dod-3), odgovora na toplinski šok (hsf-1, hsp70, hsp-16.2, hsp-4) i steroidnog signalnog puta (daf-12, fard-1, cdr-6, daf-36). Zatim sam
kvantificirala aktivnost fluorescentno obilježenih transkripcijskih faktora HSF-1 i DAF-16 te
mjerila otpornost oblića na toplinski i oksidativni stres. Međutim, promjene u ekspresiji gena,
aktivnosti DAF-16 i HSF-1, otpornost na toplinski i oksidativni stres nisu dali jasan uvid u
mehanizam produženja životnog vijeka mutanata glp-1ncl-1 s utišanim genom daf-41
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