Comparison of growth curves of mice selected and unselected for postweaning gain

Summary Mice were sampled from a line selected for increased postweaning weight gain from three to six weeks and from a randombred control line originating from the same base population. Body weights were recorded at each of 14 ages from day 5 to day 98. The Richards and logistic growth functions were fitted to the growth trajectories of each individual mouse by a generalized non-linear least squares procedure. Estimated growth parameters (asymptotic weight, rate, shape of curve, age and weight at inflection, mean absolute growth rate and mean relative growth rate) were computed for each individual. The effects of line, litter within line, sex and line × sex interactions on these estimated parameters were then studied.Both the Richards and logistic functions fitted the data equally well and the plotted trajectories coincided over most of the growth curve. There was excellent agreement between the estimates of asymptotic weight and both age and weight at inflection based on the different functions. However, both functions apparently underestimated the asymptotic weight.Analyses of the line differences showed that selection for postweaning gain increased the mean absolute growth rate over the entire curve but had no effect on the relative growth rate or the shape of the growth curve. Full-sib analyses suggested the presence of considerable genetic variation and some high genetic correlations among the estimated growth parameters.Paper number 2942 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, North Carolina. This research was supported in part by Public Health Service Research Grant GM 11546. Computing services were supported by NIH Grant FR-00011.     

Establishment of a variant cell clone growing at 41 degrees C from embryonic rat and tupaia (tree shrew) fibroblast cells

Rat and tupaia 41 degrees C temperature variant cell clones were derived from parental embryonic cells, cloned and established in tissue cultures. Both variant cell clones grew permanently at 41 degrees C. The morphology of these cell clones was altered in comparison to the original fibroblast cell clones. The cell biological characterization of the rat and tupaia 41 degrees C temperature variant cell clones showed that both cell clones were stable. After abolishing the selection pressure (incubation at 41 degrees C) for more than 10 further cell passages by incubation at 37 degrees C and then raising the temperature again to 41 degrees C, neither of the cell clones lost their newly acquired property of growing at 41 degrees C. This fact demonstrates that the newly acquired property is certain to be genetically manifest in both cell clones. The modal number of chromosomes of the rat 41 degrees C temperature variant cell clone was increased, and in the case of the tupaia variant cell clone, bimodality was observed. The plating efficiency of both cell clones did not rise significantly in comparison to the parental cells. Neither of the 41 degrees C temperature variant cell clones grew in semi-solid medium.     

The reduced nicotinamide adenine dinucleotide "oxidase" of Acholeplasma laidlawii membranes.

An NADH dehydrogenase possessing a specific activity 3-5 times that of membrane-bound enzyme was obtained by extraction of Acholeplasma laidlawii membranes with 9.0% ethanol at 43 degrees C. This dehydrogenase contained only trace amounts of iron (suggesting an uncoupled respiration), a flavin ratio of 1:2 FAD to FMN and 30-40% lipid. Its resistance to sedimentation is probably due to the high flotation density of the lipids. It efficiently utilized ferricyanide, menadione and dichlorophenol indophenol as electron acceptors, but not O2, ubiquinone Q10 or cytochrome c. Lineweaver-Burk plots of the dehydrogenase were altered to linear functions upon extraction with 9.0% ethanol. A secondary site of ferricyanide reduction could not be explained by the presence of cytochromes, which these membranes lack. In comparison to other respiratory chain-linked NADH dehydrogenases in cytochrome-containing respiratory chains, this dehydrogenase was characterized by similar Km's with ferricyanide, dichlorophenol indophenol, menadione as electron acceptors, but considerably smaller V's with ferricyanide, dichlorophenol indophenol, menadione as electron acceptors, and smaller specific activities. It was not stimulated or reactivated by the addition of FAD, FMN, Mg2+, cysteine or membrane lipids, and was less sensitive to respiratory inhibitors than unextracted enzyme. The ineffectiveness of ADP stimulation on O2 uptake, the insensitivity to oligomycin and the very low iron content of A. laidlawii membranes were considered in relation to conservation of energy by these cells. Some kinetic properties of the dehydrogenation, the uniquely high glycolipid content and apparently uncoupled respiration at Site I were noteworthy characteristics of this NADH dehydrogenase from the truncated respiratory chain of A. laidlawii.     

A case of atypical galactosemia(author's transl)]

The case of a male infant suffering from clinical manifest galactosemia in connexion with a disease of only about 70% in uridyltransferase activity is characterized as atypical galactosemia. As compared with the described "Indiana variant" of a galactosemic female infant showing an unstable uridyltransferase activity this case demonstrates an unchanged enzyme activity still after 72 hrs. The metabolic disease of this patient clearly demonstrated that the lack of a typical pronounced reduction of enzyme activity maybe not the leading criterion for excluding the clinical diagnosis of galactosemia. In the present case only the consistent galactose-free diet lead to the decisive improvement of the clinical picture. The investigation of the family revealed the typical pattern of an autosomal recessive mode of heredity.     

Estimating the potential for coral adaptation to global warming across the Indo‐West Pacific

The potential of reef‐building corals to adapt to increasing sea‐surface temperatures is often debated but has rarely been comprehensively modeled on a region‐wide scale. We used individual‐based simulations to model adaptation to warming in a coral metapopulation comprising 680 reefs and representing the whole of the Central Indo‐West Pacific. Encouragingly, some reefs—most notably Vietnam, Japan, Taiwan, New Caledonia and the southern half of the Great Barrier Reef—exhibited high capacity for adaptation and, in our model, maintained coral cover even under a rapid “business‐as‐usual” warming scenario throughout the modeled period (200 years). Higher resilience of these reefs was observed under all tested parameter settings except the models prohibiting selection and/or migration during warming. At the same time, the majority of reefs in the region tended to collapse within the first 100 years of warming. The adaptive potential (odds of maintaining high coral cover) of a given reef could be predicted based on two metrics: the reef's present‐day temperature, and the proportion of recruits immigrating from warmer locations. The latter metric explains the most variation in adaptive potential, and significantly correlates with actual coral cover changes observed throughout the region between the 1970s and the early 2000s. These findings will help prioritize coral conservation efforts and plan assisted gene flow interventions to boost the adaptive potential of specific coral populations.     

CRISPR-based DNA and RNA detection with liquid-liquid phase separation

The ability to detect specific nucleic acid sequences allows for a wide range of applications such as the identification of pathogens, clinical diagnostics, and genotyping. CRISPR-Cas proteins Cas12a and Cas13a are RNA-guided endonucleases that bind and cleave specific DNA and RNA sequences, respectively. After recognition of a target sequence, both enzymes activate indiscriminate nucleic acid cleavage, which has been exploited for sequence-specific molecular diagnostics of nucleic acids. Here, we present a label-free detection approach that uses a readout based on solution turbidity caused by liquid-liquid phase separation (LLPS). Our approach relies on the fact that the LLPS of oppositely charged polymers requires polymers to be longer than a critical length. This length dependence is predicted by the Voorn-Overbeek model, which we describe in detail and validate experimentally in mixtures of polynucleotides and polycations. We show that the turbidity resulting from LLPS can be used to detect the presence of specific nucleic acid sequences by employing the programmable CRISPR-nucleases Cas12a and Cas13a. Because LLPS of polynucleotides and polycations causes solutions to become turbid, the detection of specific nucleic acid sequences can be observed with the naked eye. We furthermore demonstrate that there is an optimal polynucleotide concentration for detection. Finally, we provide a theoretical prediction that hints towards possible improvements of an LLPS-based detection assay. The deployment of LLPS complements CRISPR-based molecular diagnostic applications and facilitates easy and low-cost nucleotide sequence detection.     

An apicoplast‐resident folate transporter is essential for sporogony of malaria parasites

Malaria parasites are fast replicating unicellular organisms and require substantial amounts of folate for DNA synthesis. Despite the central role of this critical co‐factor for parasite survival, only little is known about intraparasitic folate trafficking in Plasmodium. Here, we report on the expression, subcellular localisation and function of the parasite's folate transporter 2 (FT2) during life cycle progression in the murine malaria parasite Plasmodium berghei. Using live fluorescence microscopy of genetically engineered parasites, we demonstrate that FT2 localises to the apicoplast. In invasive P. berghei stages, a fraction of FT2 is also observed at the apical end. Upon genetic disruption of FT2, blood and liver infection, gametocyte production and mosquito colonisation remain unaltered. But in the Anopheles vector, FT2‐deficient parasites develop inflated oocysts with unusual pulp formation consisting of numerous single‐membrane vesicles, which ultimately fuse to form large cavities. Ultrastructural analysis suggests that this defect reflects aberrant sporoblast formation caused by abnormal vesicular traffic. Complete sporogony in FT2‐deficient oocysts is very rare, and mutant sporozoites fail to establish hepatocyte infection, resulting in a complete block of parasite transmission. Our findings reveal a previously unrecognised organellar folate transporter that exerts critical roles for pathogen maturation in the arthropod vector.