The in vitro culture system for spermatogonial stem cells (SSCs) is a powerful tool for exploring molecular mechanisms of male gametogenesis and gene manipulation. Very little information is ...available for fish SSC biology. Our aim was to isolate highly pure SSCs from the testis of commercially important farmed carp, Labeo rohita. The minced testis of L. rohita was dissociated with collagenase. Dissociated cells purified by two-step Ficoll gradient centrifugation followed by magnetic activated cell sorting (MACS) using Thy1.2 (CD90.2) antibody dramatically heightened recovery rate for spermatogonial cells. The purified cells were cultured in vitro conditions for more than two months in L-15 media containing 10% fetal bovine serum (FBS), 1% carp serum, and other nutrients. The proliferative cells were dividing as validated by 5-bromo-2′-deoxyuridine (BrdU) incorporation assay and formed colonies/clumps with the typical characteristics of SSCs A majority of enriched cell population represented a Vasa+, Pou5f1/pou5f1+, Ssea-1+, Tra-1-81+, plzf+, Gfrα1/gfrα1−, and c-Kit/c-kit− as detected by immunocytochemical and/or quantitative real-time polymerase chain reaction (RT-PCR) analyses. Thus, Thy1+ SSCs were enriched with greater efficiency from the mixed population of testicular cells of L. rohita. A population of enriched spermatogonial cells could be cultured in an undifferentiated state. The isolated SSCs could provide avenue for undertaking research on basic and applied reproductive biology.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
We describe a method of detecting features in retinal images using a model-based approach. The image is processed using a bank of filters in a scale space. A parametric model of the target feature is ...then proposed and the filter responses to the model calculated. A noise model is proposed, and incorporated into a maximum likelihood estimator to estimate model parameters. The estimator uses the generative parametric model to explore smoothly the scale space. This method is applied to the detection of retinal blood vessels, using a Gaussian-profiled valley as a model. A simple thresholding method is proposed as an example of using the rich estimated parameter maps to detect vessels and the results are compared against two existing vessel detectors. Our system is compared against ground truth and the output of existing systems. It is found to be comparable and, in addition, produces direct estimates of vessel calibres and contrasts. It does not use any form of region growing or vessel tracking, but thresholds a function of the estimated vessel parameters to determine vessel regions.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The prevalence of allergy is markedly low in children growing up on farms. An increasing number of studies indicate that the timing of food introduction may affect allergy development. We aimed to ...investigate if protection against allergy in farm environments may be mediated through differences in food-introduction practices between farm and non-farm families, using an explorative approach. Twenty-eight farm and 37 non-farm children were included in the FARMFLORA birth cohort. Practices of breastfeeding and introduction of formulas and complementary foods were collected by questionnaires at 6, 12, and 18 months of age. Allergy was diagnosed by pediatricians at 3 years of age. The only difference in food-introduction practices observed between farm and non-farm children was an earlier introduction of nuts in farmers (median month: 11 IQR: 8-6 in farmers, 15 12-19 in non-farmers). One farm child (4%) and 10 non-farm children (27%) were allergic at 3 years of age. Lower risk of allergy development was associated with early exclusive breastfeeding (continuous variable; OR = 0.59, 95% CI: 0.39-0.89), but also having received eggs (OR = 0.08, 95% CI: 0.13-0.54) and fish (logistic regression not applicable, P = 0.01 in likelihood ratio testing χ
2
) at 10 months of age or earlier compared to later. Our results were not affected by reverse causation, as judged by a questionnaire sent to the families in retrospect. Timing of introduction of complementary foods is unlikely to contribute to the lower risk of allergy among farm children. Although early exclusive breastfeeding was associated with a lower rate of allergy development, postponed introduction of complementary foods might increase the risk of developing allergy. Owing to the limited sample size, our results are only indicative, but support prior findings.
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DOBA, FSPLJ, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Information on the genetic structure of cultivable fish species is essential for studying molecular systematics and optimising fisheries management and fish farming. The random amplified polymorphic ...DNA (RAPD) assay was evaluated for studying genetic relationships and diversities in four species of Indian major carps (Family Cyprinidae). Thirty-four arbitrary primers were screened to identify species-specific RAPD markers among rohu (
Labeo rohita), kalbasu (
L. calbasu), catla (
Catla catla) and mrigal (
Cirrhinus mrigala). Distinct and highly reproducible RAPD profiles with a great degree of genetic variability were detected among species. On average, 45% of the scorable RAPD bands were specific to each species. Phylogenetic analysis demonstrated that kalbasu is the closest to rohu and the farthest from mrigal. Data are presented with regard to the applications of RAPD markers for hybrid identification, genetic diversity assessment and studying taxonomic relationships at a molecular level.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The effect of increase in temperature on grain yield of some winter crops (wheat, mustard, barley and chickpea) in northwest India was evaluated on the basis of historic records and through a dynamic ...crop growth model, WTGROWS. The optimal date of sowing was also evaluated in view of the increase in seasonal temperature. The yield of these crops, especially wheat, already showing signs of stagnation in most places of northwest India, is most likely to be affected by temperature changes. The solar radiation–temperature interactions study in wheat reveals some interesting trends and is seen to vary from one location to another. Keeping in view the trends in global climate change, a shift in sowing time, as an adaptation strategy is recommended. The simple and empirical relations between yield and seasonal temperature change can be well used for a crude estimate of yield dependence of temperature rise of these winter crops.
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BFBNIB, DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Asf1 (anti-silencing function 1), a well conserved protein from yeast to humans, acts as a histone chaperone and is predicted to participate in a variety of chromatin-mediated cellular processes. To ...investigate the physiological role of vertebrate Asf1 in vivo, we generated a conditional Asf1-deficient mutant from chicken DT40 cells. Induction of Asf1 depletion resulted in the accumulation of cells in S phase with decreased DNA replication and increased mitotic aberrancy forming multipolar spindles, leading to cell death. In addition, nascent chromatin in Asf1-depleted cells showed increased nuclease sensitivity, indicating impaired nucleosome assembly during DNA replication. Complementation analyses revealed that the functional domain of Asf1 for cell viability was confined to the N-terminal core domain (amino acids 1-155) that is a binding platform for histones H3/H4, CAF-1p60, and HIRA, whereas Asf1 mutant proteins, abolishing binding abilities with both p60 and HIRA, exhibit no effect on viability. These results together indicate that the vertebrate Asf1 plays a crucial role in replication-coupled chromatin assembly, cell cycle progression, and cellular viability and provide a clue of a possible role in a CAF-1- and HIRA-independent chromatin-modulating process for cell proliferation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The study was conducted in a society pond of Gandhibasti village of Abhayapuri, Bongaigaon, Assam to assess the impact of duck droppings on water quality and pond productivity. The pond was ...integrated with ducks (Anas platyrhynchos) to obtain the benefit of duck litters on the pond productivity and fish growth. The mean dry weight of 80 ducks excreta was estimated as 3.864 kg/ha/day, discharging into the pond. Indian major carps along with few exotic carp were stocked in the month of April, 2013. Supplementary feed was also given to the fishes in alternate days to ensure their steady growth. All the studied physico-chemical parameters were found within the permissible limit and the growth of fishes was also satisfactory. The maximum growth was recorded in Catla followed by silver carp, common carp, Rohu and Mrigala.
Recent advances in high throughout DNA sequencing
technologies have revolutionized for better understanding
of structure-functional relationships of genes in identifying
trait-associated ...transcriptomes and their regulated gene
expressions. Subsequent breakthroughs in gene editing
technologies such as zinc finger nucleases, transcriptional
activator-like effect or nucleases (TALEN) and CRISPR
(clustered regularly interspaced short palindromic repeats)
determined chromosomal loci so as to understand gene
functions in vivo. Such editing technologies are now being
implemented in many laboratories due to an affordable
cost and easiness of techniques. Targeted gene delivery
and disruptions are now not only restricted to standard
cell lines or stem cells, but also primary cell lines and nonmodel
agriculturally important species. Progress and
implications of gene integration and disruptions in food
fishes like salmon, carps, etc. will be highlighted. The
positive impacts on myostatin gene (negative growth
hormone regulator) disruption mediated muscular growth
have been documented. Transposon mediated gene
integration technologies for value-additions to small
indigenous aquarium fishes by expressing attractive
fluorescent color genes could be the future of rainbow
revolution. Issues linked with further-tuning with regards
to improved efficacy and specificity, while reducing offtarget
effects of gene editing tools will be addressed. There
are health and environmental concerns with genetically
modified organisms (GMOs). CRISPR/Cas9 mediated
editing generates indels and hence supposed to be free
from transgene-nontoxic and non-allergen. Scientific
progress regarding to generate genetically modified carps;
those could well be cultivated in a confinement and at the
same time economically profitable; will be highlighted.
Emphasis should be given for transfer these technologies
from the laboratory to land for the development of a
consumer-friendly sustainable farming system.
Histone acetyltransferase 1 (HAT1) is implicated for diacetylation of Lys-5 and Lys-12 of newly synthesized histone H4, the biological significance of which remains unclear. To investigate the in ...vivo role of HAT1, we generated HAT1-deficient DT40 clone (
HAT1
−/−).
HAT1
−/− cells exhibited greatly reduced diacetylation levels of Lys-5 and Lys-12, and acetylation level of Lys-5 of cytosolic and chromatin histones H4, respectively. The in vitro nucleosome assembly assay and in vivo MNase digestion assay revealed that HAT1 and diacetylation of Lys-5 and Lys-12 of histone H4 are dispensable for replication-coupled chromatin assembly.
HAT1
−/− cells had mild growth defect, conferring sensitivities to methyl methanesulfonate and camptothecin that enforce replication blocks creating DNA double strand breaks. Such heightened sensitivities were associated with prolonged late-S/G2 phase. These results indicate that HAT1 participates in recovering replication block-mediated DNA damages, probably through chromatin modulation based on acetylation of Lys-5 and Lys-12 of histone H4.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Amounts of soluble histones in cells are tightly regulated to ensure supplying them for the newly synthesized DNA and preventing the toxic effect of excess histones. Prior to incorporation into ...chromatin, newly synthesized histones H3 and H4 are highly acetylated in pre-deposition complex, wherein H4 is di-acetylated at Lys-5 and Lys-12 residues by histone acetyltransferase-1 (Hat1), but their role in histone metabolism is still unclear. Here, using chicken DT 40 cytosolic extracts, we found that histones H3/H4 and their chaperone Asf1, including RbAp48, a regulatory subunit of Hat1 enzyme, were associated with Hat1. Interestingly, in HAT1-deficient cells, cytosolic histones H3/H4 fractions on sucrose gradient centrifugation, having a sedimentation coefficient of 5–6S in DT40 cells, were shifted to lower molecular mass fractions, with Asf1. Further, sucrose gradient fractionation of semi-purified tagged Asf1-complexes showed the presence of Hat1, RbAp48 and histones H3/H4 at 5–6S fractions in the complexes. These findings suggest the possible involvement of Hat1 in regulating cytosolic H3/H4 pool mediated by Asf1-containing cytosolic H3/H4 pre-deposition complex.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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