Multiple Paternity in the Nurse Shark, Ginglymostoma Cirratum

For over a decade, we have been studying the reproductive behavior of the nurse shark, Ginglymostoma cirratum, in the Dry Torugas off the Florida Keys, an important mating and nursery ground for this species. In the course of these studies, we have used a variety of tags and tagging protocols to monitor individual animals. Here we report the use of molecular methods for the genetic analysis of nurse sharks. Specifically we have analyzed genetic variation at the MHC II alpha locus using the polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) analysis of the amplified products. We found this technique to be a relatively rapid and reliable method for identifying genetic differences between individual sharks. Applying this method to a family of sharks consisting of a mother and 32 pups, we demonstrate that at least four fathers must have fathered this brood. Multiple paternity in the nurse shark suggests a mechanism by which populations of this species may maximize genetic variability. This seems especially valuable for philopatric species whose migratory movement, and thus potential for genetic diversity, is limited.     

Interactions of aminoglycoside antibiotics with phospholipids. A deuterium nuclear magnetic resonance study.

The effect of several aminoglycoside (AG) antibiotics on aqueous multilamellar dispersions of mixtures of phosphatidylinositol (PI) and deuterated phosphatidylcholine (PC) has been studied by deuterium (2H) NMR. Isepamicin and amikacin gave rise to no significant changes in 2H-NMR lineshape relative to that of the lipid mixture without antibiotic. Both kanamycin A and B, which have a greater affinity for PI than the other two antibiotics examined in this study, induced temperature-dependent changes in 2H-NMR lineshapes and associated spectral moments. The results are consistent with an antibiotic-induced lateral phase separation giving rise to PC-enriched domains free of drug and PI-AG domains. These effects are correlated with the inhibitory potency of aminoglycosides towards PC degradation.     

Impact Loading and Locomotor-Respiratory Coordination Significantly Influence Breathing Dynamics in Running Humans

Locomotor-respiratory coupling (LRC), phase-locking between breathing and stepping rhythms, occurs in many vertebrates. When quadrupedal mammals gallop, 1∶1 stride per breath coupling is necessitated by pronounced mechanical interactions between locomotion and ventilation. Humans show more flexibility in breathing patterns during locomotion, using LRC ratios of 2∶1, 2.5∶1, 3∶1, or 4∶1 and sometimes no coupling. Previous studies provide conflicting evidence on the mechanical significance of LRC in running humans. Some studies suggest LRC improves breathing efficiency, but others suggest LRC is mechanically insignificant because ‘step-driven flows’ (ventilatory flows attributable to step-induced forces) contribute a negligible fraction of tidal volume. Yet, although step-driven flows are brief, they cause large fluctuations in ventilatory flow. Here we test the hypothesis that running humans use LRC to minimize antagonistic effects of step-driven flows on breathing. We measured locomotor-ventilatory dynamics in 14 subjects running at a self-selected speed (2.6±0.1 ms⁻¹) and compared breathing dynamics in their naturally ‘preferred’ and ‘avoided’ entrainment patterns. Step-driven flows occurred at 1-2X step frequency with peak magnitudes of 0.97±0.45 Ls⁻¹ (mean ±S.D). Step-driven flows varied depending on ventilatory state (high versus low lung volume), suggesting state-dependent changes in compliance and damping of thoraco-abdominal tissues. Subjects naturally preferred LRC patterns that minimized antagonistic interactions and aligned ventilatory transitions with assistive phases of the step. Ventilatory transitions initiated in ‘preferred’ phases within the step cycle occurred 2x faster than those in ‘avoided’ phases. We hypothesize that humans coordinate breathing and locomotion to minimize antagonistic loading of respiratory muscles, reduce work of breathing and minimize rate of fatigue. Future work could address the potential consequences of locomotor-ventilatory interactions for elite endurance athletes and individuals who are overweight or obese, populations in which respiratory muscle fatigue can be limiting.     

Partial migration of the nurse shark, <Emphasis Type="Italic">Ginglymostoma cirratum</Emphasis> (Bonnaterre), from the Dry Tortugas Islands

Nurse sharks have not previously been known to migrate. Nurse sharks of the Dry Tortugas (DRTO) mating population have a highly predictable periodic residency cycle, returning to the Dry Tortugas Courtship and Mating Ground (DTCMG) annually (males) or bi- to triennially (females) during the June/July mating season. For 23 years we have followed the movements of 76 recaptured adults of a total of 115 tagged adults. Telemetry detections of 40 females tagged with acoustic transmitters show that most tagged and presumably post-partum females are continuously present in the DRTO in the fall, winter and early spring following the June mating season but these females depart in late March to early May. Detections reveal these females avoid the DTCMG completely during the next mating season, returning from late summer to fall. Telemetry records of nine of 17 adult males that co-habited with these females in the DTCMG depart DRTO waters every July. Both sexes may overwinter in the DRTO. Between 2011 and 2016 three males and five females with transmitters were detected to move up the west coast of Florida outside of the mating season as far north as the waters off Tampa Bay (335 km). Six others were only detected in the lower Florida Keys (292 km). Nine sharks returned to DRTO; one returned six times. Some overwintered and some resumed courtship in June, demonstrating both resident and migratory contingents within their population, partial migration and an ability to navigate with high spatial and temporal precision.     

Sucrose requirements and lipid utilization during germination of interior spruce (Picea glauca engelmannii complex) somatic embryos

Both somatic and excised zygotic embryos of interior spruce (Picea glauca engelmannii complex) required exogenous sucrose in the medium for germination in vitro. Over a period of 29 days on sucrose-containing medium germinants with roots and epicotyls developed from both kinds of embryo, and their content of linolenic acid (9,12,15-18:3) increased about six- to eightfold. Without added sucrose, embryos showed retarded growth or were necrotic, and the content of linolenic acid was barely detectable in their fatty acid profiles. Through¹⁴C-sucrose uptake studies, it was determined that germinants consumed only 25% of the sucrose available in a 1% (wt/vol) sucrose-containing medium. Since no radiolabelled fatty acids were detected, it appears that externally supplied sucrose was not used in the synthesis of lipids. Although sucrose was present during plantlet development, 72% of the initial lipids were consumed. To some extent, the plantlets appeared to be obligate storage lipid utilizers.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - FAMEs Fatty acid methyl esters - HPLC High-performance liquid chromatography     

Endonuclease from Micrococcus luteus Which Has Activity Toward Ultraviolet-Irradiated Deoxyribonucleic Acid: Purification and Properties

An endonuclease purified approximately 3,200-fold from Micrococcus luteus is active on native ultraviolet-irradiated deoxyribonucleic acid (DNA), but is inactive on unirradiated native or denatured DNA and has no activity toward irradiated denatured DNA. The major type of lesion for the nucleolytic activity is the cyclobutane pyrimidine dimer. The enzyme makes a number of single-strand breaks approximately equal to the number of dimers, but dimers are not excised. This endonuclease-a small molecular weight protein-therefore has all the attributes hypothesized for the first enzyme in the sequential steps in repair of DNA in vivo. Another paper shows that the endonuclease is able to reactivate ultraviolet-irradiated transforming DNA.     

The binding of auxin to the Arabidopsis auxin influx transporter AUX1

The cellular import of the hormone auxin is a fundamental requirement for the generation of auxin gradients that control a multitude of plant developmental processes. The AUX/LAX family of auxin importers, exemplified by AUX1 from Arabidopsis (Arabidopsis thaliana), has been shown to mediate auxin import when expressed heterologously. The quantitative nature of the interaction between AUX1 and its transport substrate indole-3-acetic acid (IAA) is incompletely understood, and we sought to address this in the present investigation. We expressed AUX1 to high levels in a baculovirus expression system and prepared membrane fragments from baculovirus-infected insect cells. These membranes proved suitable for determination of the binding of IAA to AUX1 and enabled us to determine a K(d) of 2.6 mum, comparable with estimates for the K(m) for IAA transport. The efficacy of a number of auxin analogues and auxin transport inhibitors to displace IAA binding from AUX1 has also been determined and can be rationalized in terms of their physiological effects. Determination of the parameters describing the initial interaction between a plant transporter and its hormone ligand provides novel quantitative data for modeling auxin fluxes.     

The PTEN phosphatase controls intestinal epithelial cell polarity and barrier function: role in colorectal cancer progression

Background

The PTEN phosphatase acts on phosphatidylinositol 3,4,5-triphosphates resulting from phosphatidylinositol 3-kinase (PI3K) activation. PTEN expression has been shown to be decreased in colorectal cancer. Little is known however as to the specific cellular role of PTEN in human intestinal epithelial cells. The aim of this study was to investigate the role of PTEN in human colorectal cancer cells.

Methodology/Principal Findings

Caco-2/15, HCT116 and CT26 cells were infected with recombinant lentiviruses expressing a shRNA specifically designed to knock-down PTEN. The impact of PTEN downregulation was analyzed on cell polarization and differentiation, intercellular junction integrity (expression of cell-cell adhesion proteins, barrier function), migration (wound assay), invasion (matrigel-coated transwells) and on tumor and metastasis formation in mice. Electron microscopy analysis showed that lentiviral infection of PTEN shRNA significantly inhibited Caco-2/15 cell polarization, functional differentiation and brush border development. A strong reduction in claudin 1, 3, 4 and 8 was also observed as well as a decrease in transepithelial resistance. Loss of PTEN expression increased the spreading, migration and invasion capacities of colorectal cancer cells in vitro. PTEN downregulation also increased tumor size following subcutaneous injection of colorectal cancer cells in nude mice. Finally, loss of PTEN expression in HCT116 and CT26, but not in Caco-2/15, led to an increase in their metastatic potential following tail-vein injections in mice.

Conclusions/Significance

Altogether, these results indicate that PTEN controls cellular polarity, establishment of cell-cell junctions, paracellular permeability, migration and tumorigenic/metastatic potential of human colorectal cancer cells.     

Integrated gut/liver microphysiological systems elucidates inflammatory inter‐tissue crosstalk

A capability for analyzing complex cellular communication among tissues is important in drug discovery and development, and in vitro technologies for doing so are required for human applications. A prominent instance is communication between the gut and the liver, whereby perturbations of one tissue can influence behavior of the other. Here, we present a study on human gut‐liver tissue interactions under normal and inflammatory contexts, via an integrative multi‐organ platform comprising human liver (hepatocytes and Kupffer cells), and intestinal (enterocytes, goblet cells, and dendritic cells) models. Our results demonstrated long‐term (>2 weeks) maintenance of intestinal (e.g., barrier integrity) and hepatic (e.g., albumin) functions in baseline interaction. Gene expression data comparing liver in interaction with gut, versus isolation, revealed modulation of bile acid metabolism. Intestinal FGF19 secretion and associated inhibition of hepatic CYP7A1 expression provided evidence of physiologically relevant gut‐liver crosstalk. Moreover, significant non‐linear modulation of cytokine responses was observed under inflammatory gut‐liver interaction; for example, production of CXCR3 ligands (CXCL9,10,11) was synergistically enhanced. RNA‐seq analysis revealed significant upregulation of IFNα/β/γ signaling during inflammatory gut‐liver crosstalk, with these pathways implicated in the synergistic CXCR3 chemokine production. Exacerbated inflammatory response in gut‐liver interaction also negatively affected tissue‐specific functions (e.g., liver metabolism). These findings illustrate how an integrated multi‐tissue platform can generate insights useful for understanding complex pathophysiological processes such as inflammatory organ crosstalk. Biotechnol. Bioeng. 2017;114: 2648–2659. © 2017 Wiley Periodicals, Inc.