We developed a simple and reliable method for the isolation of haploid nuclei from fresh and frozen testes. The described protocol uses readily available reagents in combination with flow cytometry ...to separate haploid and diploid nuclei. The protocol can be completed within 1 hour and the resulting individual haploid nuclei have intact morphology. The isolated nuclei are suitable for library preparation for high-throughput DNA and RNA sequencing using bulk or single nuclei. The protocol was optimised with mouse testes and we anticipate that it can be applied for the isolation of mature sperm from other mammals including humans.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We developed a simple and reliable method for the isolation of haploid nuclei from fresh and frozen testes. The described protocol uses readily available reagents in combination with flow cytometry ...to separate haploid and diploid nuclei. The protocol can be completed within 1 hour and the resulting individual haploid nuclei have intact morphology. The isolated nuclei are suitable for library preparation for high-throughput DNA and RNA sequencing using bulk or single nuclei. The protocol was optimised with mouse testes and we anticipate that it can be applied for the isolation of mature sperm from other mammals including humans.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The interactions of mitochondria with the endoplasmic reticulum (ER) are crucial for maintaining proper mitochondrial morphology, function and dynamics. This enables cells to utilize their ...mitochondria optimally for energy production and anabolism, and it further provides for metabolic control over developmental decisions. In fungi, a key mechanism by which ER and mitochondria interact is via a membrane tether, the protein complex ERMES (ER-Mitochondria Encounter Structure). In the model yeast
, the mitochondrial GTPase Gem1 interacts with ERMES, and it has been proposed to regulate its activity. Here we report on the first characterization of Gem1 in a human fungal pathogen. We show that in
Gem1 has a dominant role in ensuring proper mitochondrial morphology, and our data is consistent with Gem1 working with ERMES in this role. Mitochondrial respiration and steady state cellular phospholipid homeostasis are not impacted by inactivation of
in
. There are two major virulence-related consequences of disrupting mitochondrial morphology by
inactivation:
becomes hypersusceptible to cell wall stress, and is unable to grow invasively. In the
Δ
Δ mutant, it is specifically the invasive capacity of hyphae that is compromised, not the ability to transition from yeast to hyphal morphology, and this phenotype is shared with ERMES mutants. As a consequence of the hyphal invasion defect, the
Δ
Δ mutant is drastically hypovirulent in the worm infection model. Activation of the mitogen activated protein (MAP) kinase Cek1 is reduced in the
Δ
Δ mutant, and this function could explain both the susceptibility to cell wall stress and lack of invasive growth. This result establishes a new, respiration-independent mechanism of mitochondrial control over stress signaling and hyphal functions in
. We propose that ER-mitochondria interactions and the ER-Mitochondria Organizing Network (ERMIONE) play important roles in adaptive responses in fungi, in particular cell surface-related mechanisms that drive invasive growth and stress responsive behaviors that support fungal pathogenicity.
RIO protein kinases (RIOKs) are a relatively conserved family of enzymes implicated in cell cycle control and ribosomal RNA processing. Despite their functional importance, they remain a poorly ...understood group of kinases in multicellular organisms. Here, we show that the C. elegans genome contains one member of each of the three RIOK sub-families and that each of the genes coding for them has a unique tissue expression pattern. Our analysis showed that the gene encoding RIOK-1 (riok-1) was broadly and strongly expressed. Interestingly, the intestinal expression of riok-1 was dependent upon two putative binding sites for the oxidative and xenobiotic stress response transcription factor SKN-1. RNA interference (RNAi)-mediated knock down of riok-1 resulted in germline defects, including defects in germ line stem cell proliferation, oocyte maturation and the production of endomitotic oocytes. Taken together, our findings indicate new functions for RIOK-1 in post mitotic tissues and in reproduction.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Adequate dietary intake of essential metals such as zinc is important for maintaining homeostasis. Abnormal zinc intake in Caenorhabditis elegans has been shown to increase or decrease normal ...lifespan by influencing the insulin/IGF‐1 pathway. Distribution of zinc is achieved by a family of highly conserved zinc transport proteins (ZIPT in C. elegans). This study investigated the role of the zipt family of genes and showed that depletion of individual zipt genes results in a decreased lifespan. Moreover, zipt‐16 and zipt‐17 mutants synthetically interact with the insulin/IGF cofactors daf‐16 and skn‐1, and cause abnormal localisation of DAF‐16. This study suggests that the zipt family of genes are required for maintaining normal lifespan through influencing the insulin/IGF‐1 pathway.
Meiotic crossovers are required for accurate chromosome segregation and producing new allelic combinations. Meiotic crossover numbers are tightly regulated within a narrow range, despite an excess of ...initiating DNA double-strand breaks. Here, we reveal the tumor suppressor FANCM as a meiotic anti-crossover factor in mammals. We use unique large-scale crossover analyses with both single-gamete sequencing and pedigree-based bulk-sequencing datasets to identify a genome-wide increase in crossover frequencies in Fancm-deficient mice. Gametogenesis is heavily perturbed in Fancm loss-of-function mice, which is consistent with the reproductive defects reported in humans with biallelic FANCM mutations. A portion of the gametogenesis defects can be attributed to the cGAS-STING pathway after birth. Despite the gametogenesis phenotypes in Fancm mutants, both sexes are capable of producing offspring. We propose that the anti-crossover function and role in gametogenesis of Fancm are separable and will inform diagnostic pathways for human genomic instability disorders.
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•FancmΔ2/Δ2 limits meiotic crossovers in mammals and crossover interference is unaffected•Despite dramatic defects in gametogenesis, FancmΔ2/Δ2 mice can produce live offspring•FancmΔ2/Δ2 mice are sub-fertile in a strain-dependent manner•cGAS-STING drives spermatogonial defects in FancmΔ2/Δ2 mice
Tsui and Lyu et al. demonstrate dual roles of Fancm in mice using a combination of wet- and dry-lab techniques. They show that Fancm-deficient mice have significantly increased meiotic crossovers but are still able to generate offspring despite perturbed gametogenesis.
In 1994 the North American Free Trade Agreement (NAFTA) was signed between the United States, Canada, and Mexico. The pact’s purpose was to eliminate trade barriers in order to enhance economic ...interactions between the three parties. This paper intends to gauge the effect of NAFTA on agricultural production and farmer well-being in the Southeastern portion of the United States, specifically for smaller farms (499 acres or less). Over the last twenty years farm consolidation and a trade deficit have developed in the fruit and produce sector for the United States which may be related to NAFTA. Other crops, specifically the grain commodities, however, have benefited from industry growth. Using a novel dataset covering all fifty US states in the time span between 1992 and 2007, this paper compares Midwestern commodity producing states and industries against Southeastern predominately fruit and vegetable growers pre and post-NAFTA. It is apparent from the data gathered there are some stark differences between the two regions. Results infer that the exogenous effect of NAFTA has corresponded with both consolidation of farms and the reduction in percentage of smaller farms in the Southeast. From a policy perspective, my findings imply that trade deals such as NAFTA lead to redistributive outcomes and policymakers should consider ways to support those adversely affected.
The study presents a mathematical model of Bruk Temkin-Gorodsky Novakovic (BT-GN) reaction system employing palladium-catalyzed oscillatory carbonylation reaction of mono alkyne-terminated ...poly(ethylene glycol) methyl ether. The proposed model advances simulation capabilities of this particular chemical oscillator but also BT-GN reactions in general. The model was verified using experimental data where good agreement is achieved and existence of complex pH oscillations like burst oscillations was matched. Furthermore, the model was used to predict reaction conditions capable of producing further complexities and initial conditions that would lead to desired period of pH oscillations. Having such a powerful tool, enhances our capabilities to study and further develop BT-GN oscillators with a reduced experimental effort.
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