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In gene therapy, potential integration of therapeutic transgene into host cell genomes is a serious risk that can lead to insertional mutagenesis and tumorigenesis. Viral vectors are often used as the gene delivery vehicle, but they are prone to undergoing integration events. More recently, non-viral delivery of linear DNAs having modified geometry such as closed-end linear duplex DNA (CELiD) have shown promise as an alternative, due to prolonged transgene expression and less cytotoxicity. However, whether modified-end linear DNAs can also provide a safe, non-integrating gene transfer remains unanswered. Herein, we compare the genomic integration frequency upon transfection of cells with expression vectors in the forms of circular plasmid, unmodified linear DNA, CELiDs with thioester loops, and Streptavidin-conjugated blocked-end linear DNA. All of the forms of linear DNA resulted in a high fraction of the cells being stably transfected-between 10 and 20% of the initially transfected cells. These results indicate that blocking the ends of linear DNA is insufficient to prevent integration., (© 2023. The Author(s).)

The spectacular fossil fauna and flora preserved in the Upper Cretaceous terrestrial strata of North America's Western Interior Basin record an exceptional peak in the diversification of fossil vertebrates in the Campanian, which has been termed the 'zenith of dinosaur diversity'. The wide latitudinal distribution of rocks and fossils that represent this episode, spanning from northern Mexico to the northern slopes of Alaska, provides a unique opportunity to gain insights into dinosaur paleoecology and to address outstanding questions regarding faunal provinciality in connection to paleogeography and climate. Whereas reliable basin-wide correlations are fundamental to investigations of this sort, three decades of radioisotope geochronology of various vintages and limited compatibility has complicated correlation of distant fossil-bearing successions and given rise to contradictory paleobiogeographic and evolutionary hypotheses. Here we present new U-Pb geochronology by the CA-ID-TIMS method for 16 stratigraphically well constrained bentonite beds, ranging in age from 82.419 ± 0.074 Ma to 73.496 ± 0.039 Ma (2σ internal uncertainties), and the resulting Bayesian age models for six key fossil-bearing formations over a 1600 km latitudinal distance from northwest New Mexico, USA to southern Alberta, Canada. Our high-resolution chronostratigraphic framework for the upper Campanian of the Western Interior Basin reveals that despite their contrasting depositional settings and basin evolution histories, significant age overlap exists between the main fossil-bearing intervals of the Kaiparowits Formation (southern Utah), Judith River Formation (central Montana), Two Medicine Formation (western Montana) and Dinosaur Park Formation (southern Alberta). Pending more extensive paleontologic collecting that would allow more rigorous faunal analyses, our results support a first-order connection between paleoecologic and fossil diversities and help overcome the chronostratigraphic ambiguities that have impeded the testing of proposed models of latitudinal provinciality of dinosaur taxa during the Campanian., (© 2022. The Author(s).)

Afro-Eurasian monkeys originated in the Miocene and are the most species-rich modern primate family. Molecular and fossil data have provided considerable insight into their evolutionary divergence, but we know considerably less about the evolutionary processes that underlie these differences. Here, we apply tests developed from quantitative genetics theory to a large (n > 3000) cranio-mandibular morphometric dataset, investigating the relative importance of adaptation (natural selection) and neutral processes (genetic drift) in shaping diversity at different taxonomic levels, an approach applied previously to monkeys of the Americas, apes, hominins, and other vertebrate taxa. Results indicate that natural selection, particularly for differences in size, plays a significant role in diversifying Afro-Eurasian monkeys as a whole. However, drift appears to better explain skull divergence within the subfamily Colobinae, and in particular the African colobine clade, likely due to habitat fragmentation. Small and declining population sizes make it likely that drift will continue in this taxon, with potentially dire implications for genetic diversity and future resilience in the face of environmental change. For the other taxa, many of whom also have decreasing populations and are threatened, understanding adaptive pressures similarly helps identify relative vulnerability and may assist with prioritising scarce conservation resources., (© 2022. The Author(s).)

Calcium (Ca 2+ ) is well known as a second messenger in eukaryotes, where Ca 2+ signaling controls life-sustaining cellular processes. Although bacteria produce the components required for Ca 2+ signaling, little is known about the mechanisms of bacterial Ca 2+ signaling. Previously, we have identified a putative Ca 2+ -binding protein EfhP (PA4107) with two canonical EF-hand motifs and reported that EfhP mediates Ca 2+ regulation of virulence factors production and infectivity in Pseudomonas aeruginosa, a human pathogen causing life-threatening infections. Here, we show that EfhP selectively binds Ca 2+ with 13.7 µM affinity, and that mutations at the +X and -Z positions within each or both EF-hand motifs abolished Ca 2+ binding. We also show that the hydrophobicity of EfhP increased in a Ca 2+ -dependent manner, however no such response was detected in the mutated proteins. 15  N-NMR showed Ca 2+ -dependent chemical shifts in EfhP confirming Ca 2+ -binding triggered structural rearrangements in the protein. Deletion of efhP impaired P. aeruginosa survival in macrophages and virulence in vivo. Disabling EfhP Ca 2+ binding abolished Ca 2+ induction of pyocyanin production in vitro. These data confirm that EfhP selectively binds Ca 2+ , which triggers its structural changes required for the Ca 2+ regulation of P. aeruginosa virulence, thus establishing the role of EfhP as a Ca 2+ sensor., (© 2022. The Author(s).)

Mesozoic birds display considerable diversity in size, flight adaptations and feather organization 1-4 , but exhibit relatively conserved patterns of beak shape and development 5-7 . Although Neornithine (that is, crown group) birds also exhibit constraint on facial development 8,9 , they have comparatively diverse beak morphologies associated with a range of feeding and behavioural ecologies, in contrast to Mesozoic birds. Here we describe a crow-sized stem bird, Falcatakely forsterae gen. et sp. nov., from the Late Cretaceous epoch of Madagascar that possesses a long and deep rostrum, an expression of beak morphology that was previously unknown among Mesozoic birds and is superficially similar to that of a variety of crown-group birds (for example, toucans). The rostrum of Falcatakely is composed of an expansive edentulous maxilla and a small tooth-bearing premaxilla. Morphometric analyses of individual bony elements and three-dimensional rostrum shape reveal the development of a neornithine-like facial anatomy despite the retention of a maxilla-premaxilla organization that is similar to that of nonavialan theropods. The patterning and increased height of the rostrum in Falcatakely reveals a degree of developmental lability and increased morphological disparity that was previously unknown in early branching avialans. Expression of this phenotype (and presumed ecology) in a stem bird underscores that consolidation to the neornithine-like, premaxilla-dominated rostrum was not an evolutionary prerequisite for beak enlargement.

The fossil record of mammaliaforms (mammals and their closest relatives) of the Mesozoic era from the southern supercontinent Gondwana is far less extensive than that from its northern counterpart, Laurasia 1,2 . Among Mesozoic mammaliaforms, Gondwanatheria is one of the most poorly known clades, previously represented by only a single cranium and isolated jaws and teeth 1-5 . As a result, the anatomy, palaeobiology and phylogenetic relationships of gondwanatherians remain unclear. Here we report the discovery of an articulated and very well-preserved skeleton of a gondwanatherian of the latest age (72.1-66 million years ago) of the Cretaceous period from Madagascar that we assign to a new genus and species, Adalatherium hui. To our knowledge, the specimen is the most complete skeleton of a Gondwanan Mesozoic mammaliaform that has been found, and includes the only postcranial material and ascending ramus of the dentary known for any gondwanatherian. A phylogenetic analysis including the new taxon recovers Gondwanatheria as the sister group to Multituberculata. The skeleton, which represents one of the largest of the Gondwanan Mesozoic mammaliaforms, is particularly notable for exhibiting many unique features in combination with features that are convergent on those of therian mammals. This uniqueness is consistent with a lineage history for A. hui of isolation on Madagascar for more than 20 million years.

Previously known only from isolated teeth and lower jaw fragments recovered from the Cretaceous and Palaeogene of the Southern Hemisphere, the Gondwanatheria constitute the most poorly known of all major mammaliaform radiations. Here we report the discovery of the first skull material of a gondwanatherian, a complete and well-preserved cranium from Upper Cretaceous strata in Madagascar that we assign to a new genus and species. Phylogenetic analysis strongly supports its placement within Gondwanatheria, which are recognized as monophyletic and closely related to multituberculates, an evolutionarily successful clade of Mesozoic mammals known almost exclusively from the Northern Hemisphere. The new taxon is the largest known mammaliaform from the Mesozoic of Gondwana. Its craniofacial anatomy reveals that it was herbivorous, large-eyed and agile, with well-developed high-frequency hearing and a keen sense of smell. The cranium exhibits a mosaic of primitive and derived features, the disparity of which is extreme and probably reflective of a long evolutionary history in geographic isolation.

Many lines of evidence have been brought to bear on the question of theropod feeding ecology, including functional and physiological considerations, morphological constraints, taphonomic associations, and telling--although rare--indications of direct ingestion. Tooth marks of theropods, although rarely described and generally left unassigned to a particular taxon, can provide unique clues into predator-prey interaction, and can also yield insights into the extent of carcass utilization. Here we describe a sample of tooth-marked dinosaur bone recovered from three well-documented localities in the Upper Cretaceous Maevarano Formation of Madagascar that provides insights into the feeding ecology of the abelisaurid theropod Majungatholus atopus. Intensely tooth-marked elements from multiple individuals show that Majungatholus defleshed dinosaur carcasses. Furthermore, Majungatholus clearly fed upon the remains of not only sauropods, but also conspecifics, and thus was a cannibal. Cannibalism is a common ecological strategy among extant carnivores, but until now the evidence in relation to carnivorous dinosaurs has been sparse and anecdotal.

During the early months of life, gains in length and weight are more rapid by formula-fed than by breast-fed infants and we and others have speculated that the greater gains of the formula-fed infants are the result of greater food intake. If overfeeding during early infancy resulted in establishment of habits of overeating, or if, for any other reason, diet-induced fatness in infancy persisted into childhood, we might be able to demonstrate differences in fatness in childhood related to mode of feeding (breast or bottle) during infancy. We therefore examined at age 8 years 469 children born in 1966-1971 who had been studied intensely in our unit from 8 to 112 days of age. At age 8 years there were no differences in indices of fatness related to mode of feeding during infancy. Serum concentrations of cholesterol at age 8 years were also of interest because of reports from animal studies that differences in feeding during early life may be responsible for subsequent differences in cholesterol homeostasis. Cholesterol concentrations at age 8 years did not demonstrate significant differences related to mode of feeding during infancy. It is possible, however, that age 8 years is too early for an effect to be demonstrated.

The least abundant stable isotope of iron, 58Fe (natural abundance 0.322 weight %), was administered orally to infants to explore the feasibility of using a stable rather than a radioisotope in studies of iron absorption. The dose of 58Fe was given between feedings at age 126 days. The mass isotope ratio, 58Fe/57Fe, was determined in blood by inductively coupled plasma mass spectroscopy and at ages 140, 168, and 196 days. The percentage of the 58Fe dose entering the circulation (3.2 to 16.0%) was inversely correlated with serum ferritin concentration (r = -0.867, p less than 0.01). For individual infants the SD of the percentage of administered dose of iron appearing in the circulation ranged from 0.22 to 1.28. We conclude that the method is likely to be suitable for within-subject comparisons of iron availability from foods. Because of the large between-subject variation, we are pessimistic for this age group about the usefulness of study designs based on group comparisons.

To determine the bioavailability of iron from iron-fortified infant foods, we have determined erythrocyte incorporation of the stable isotope, 58Fe, after feeding the following foods extrinsically labeled with 58Fe: 1) rice cereal with apples and bananas ("cereal-fruit product"), 2) Mead Johnson Enriched Baby Food (MJEBF), a vitamin, mineral, and protein-enriched rice cereal, 3) vegetables and beef ("vegetable-beef product"), 4) grape-juice, and 5) MJEBF. Foods 1-4 were fortified with ferrous sulfate, and food 5 was fortified with ferrous fumarate. Blood was obtained at ages 140, 168, and 196 d of age, and the test meal was fed under standardized conditions at 154 d of age. Erythrocyte incorporation of the 58Fe label was determined from the increase in the mass isotope ratio, 58Fe/57Fe, from the baseline value (at 140 d of age) to the follow-up values. The mass isotope ratio was determined by inductively coupled mass spectrometry. Geometric mean total iron incorporation into erythrocytes from the test meal of MJEBF fortified with ferrous sulfate (food 2) was 0.05 mg, and from the vegetable-beef product test meal (food 3) was 0.08 mg. The low value for MJEBF is presumably explained by the low level of iron fortification. The low value for the vegetable-beef product may reflect the presence of inhibitors of iron absorption. Geometric mean erythrocyte incorporations of iron from the test meals with foods 1, 4 and 5 were 0.15, 0.14, and 0.18 mg, respectively. These erythrocyte incorporation values are 20 to 26% of the estimated 0.7 mg requirement for absorbed iron, and therefore seem nutritionally important.

Because of reluctance to use radioisotopes for studies of iron absorption in children, we have explored the feasibility of using the least abundant stable isotope of iron, 58Fe (natural abundance, 0.322 weight %) in a study of nonheme iron absorption. With a balanced cross-over design, each of 16 school-age children was fed a standardized lunch on 3 consecutive days and, 28 days later, an alternate standardized lunch on 3 consecutive days. The lunch included either a beef patty or a beef-soy patty. The mass isotope ratio, 58Fe/57Fe (MIR58/57), was measured in blood by inductively coupled plasma mass spectroscopy before and 14 days after (i.e. study day 15) consuming the three lunches. The MIR58/57 on study day 15 was used as a baseline value for lunches fed on study days 29, 30, and 31. Incorporation of 58Fe into erythrocytes was greater from the lunch with beef patty than from the lunch with beef-soy patty (geometric mean values 2.02 and 1.05% of the dose, p less than 0.03). Based on the similarity of our results with those obtained in adults with radioisotopes, we conclude that 58Fe is a satisfactory tag for studies of nonheme iron absorption from meals.