Colonization history of Galapagos giant tortoises: Insights from mitogenomes support the progression rule

Galapagos giant tortoises (Chelonoidis spp.) are a group of large, long-lived reptiles that includes 14 species, 11 of which are extant and threatened by human activities and introductions of non-native species. Here, we evaluated the phylogenetic relationships of all extant and two extinct species (Chelonoidis abingdonii from the island of Pinta and Chelonoidis niger from the island of Floreana) using Bayesian and maximum likelihood analysis of complete or nearly complete mitochondrial genomes. We also provide an updated phylogeographic scenario of their colonization of the Galapagos Islands using chrono-phylogenetic and biogeographic approaches. The resulting phylogenetic trees show three major groups of species: one from the southern, central, and western Galapagos Islands; the second from the northwestern islands; and the third group from the northern, central, and eastern Galapagos Islands. The time-calibrated phylogenetic and ancestral area reconstructions generally align with the geologic ages of the islands. The divergence of the Galapagos giant tortoises from their South American ancestor likely occurred in the upper Miocene. Their diversification on the Galapagos adheres to the island progression rule, starting in the Pleistocene with the dispersal of the ancestral form from the two oldest islands (San Cristóbal and Española) to Santa Cruz, Santiago, and Pinta, followed by multiple colonizations from different sources within the archipelago. Our work provides an example of how to reconstruct the history of endangered taxa in spite of extinctions and human-mediated dispersal events and provides a framework for evaluating the contribution of colonization and in situ speciation to the diversity of other Galapagos lineages.     

Neuroendocrine effects of light

The light/dark cycle to which animals, and possibly humans, are exposed has a major impact on their physiology. The mechanisms whereby specific tissues respond to the light/dark cycle involve the pineal hormone melatonin. The pineal gland, an end organ of the visual system in mammals, produces the hormone melatonin only at night, at which time it is released into the blood. The duration of elevated nightly melatonin provides every tissue with information about the time of day and time of year (in animals that are kept under naturally changing photoperiods). Besides its release in a circadian mode, melatonin is also discharged in a pulsatile manner; the physiological significance, if any, of pulsatile melatonin release remains unknown. The exposure of animals including man to light at night rapidly depresses pineal melatonin synthesis and, therefore, blood melatonin levels drop precipitously. The brightness of light at night required to depress melatonin production is highly species specific. In general, the pineal gland of nocturnally active mammals, which possess rod-dominated retinas, is more sensitive to inhibition by light than is the pineal gland of diurnally active animals (with cone-dominated retinas). Because of the ability of the light/dark cycle to determine melatonin production, the photoperiod is capable of influencing the function of a variety of endocrine and non-endocrine organs. Indeed, melatonin is a ubiquitously acting pineal hormone with its effects on the neuroendocrine system having been most thoroughly investigated. Thus, in nonhuman photoperiodic mammals melatonin regulates seasonal reproduction; in humans also, the indole has been implicated in the control of reproductive physiology.Summary of a Plenary Lecture presented by the author in Vienna, August, 1990     

Physiologically based pharmacokinetic/pharmacodynamic model for the prediction of morphine brain disposition and analgesia in adults and children

Morphine is a widely used opioid analgesic, which shows large differences in clinical response in children, even when aiming for equivalent plasma drug concentrations. Age-dependent brain disposition of morphine could contribute to this variability, as developmental increase in blood-brain barrier (BBB) P-glycoprotein (Pgp) expression has been reported. In addition, age-related pharmacodynamics might also explain the variability in effect. To assess the influence of these processes on morphine effectiveness, a multi-compartment brain physiologically based pharmacokinetic/pharmacodynamic (PB-PK/PD) model was developed in R (Version 3.6.2). Active Pgp-mediated morphine transport was measured in MDCKII-Pgp cells grown on transwell filters and translated by an in vitro-in vivo extrapolation approach, which included developmental Pgp expression. Passive BBB permeability of morphine and its active metabolite morphine-6-glucuronide (M6G) and their pharmacodynamic parameters were derived from experiments reported in literature. Model simulations after single dose morphine were compared with measured and published concentrations of morphine and M6G in plasma, brain extracellular fluid (ECF) and cerebrospinal fluid (CSF), as well as published drug responses in children (1 day– 16 years) and adults. Visual predictive checks indicated acceptable overlays between simulated and measured morphine and M6G concentration-time profiles and prediction errors were between 1 and -1. Incorporation of active Pgp-mediated BBB transport into the PB-PK/PD model resulted in a 1.3-fold reduced brain exposure in adults, indicating only a modest contribution on brain disposition. Analgesic effect-time profiles could be described reasonably well for older children and adults, but were largely underpredicted for neonates. In summary, an age-appropriate morphine PB-PK/PD model was developed for the prediction of brain pharmacokinetics and analgesic effects. In the neonatal population, pharmacodynamic characteristics, but not brain drug disposition, appear to be altered compared to adults and older children, which may explain the reported differences in analgesic effect.     

Ontogeny of pituitary cell-types and the hypothalamo-hypophysial relationship during early development of chum salmon,Oncorhynchus keta

Tissue non-specific alkaline phosphatase is a membrane-bound glycoprotein enzyme which is characterized by its phosphohydrolytic, protein phosphatase, and phosphotransferase activities. This enzyme is distributed virtually in all mammalian tissues, particularly during embryonic development. Its expression is stagespecific and can be demonstrated in the developing embryo as early as the 2-cell stage. It has been suggested that tissue non-specific alkaline phosphatase might play a role in tissue formation. In the study reported here, a genetransfer approach was employed to investigate possible roles for this enzyme by inserting the cDNA for rat tissue non-specific alkaline phosphatase into CHO and LLC-PK1 cells. Permanently transfected cell-lines expressing varying levels of alkaline phosphatase were estblished. The data showed that functional enzyme was expressed in the transfected cells. Cell spreading and attachment were enhanced in transfected CHO cells expressing high levels of tissue non-specific alkaline phosphatase but not in the LLC-PK1 cells. Further, in CHO cells, proliferation was shown to be inversely proportional to the level of the tissue non-specific alkaline phosphatase expression. Homotypic cell association was demonstrated in both alkaline phosphatase-positive and alkaline phosphatase-negative cells in both CHO and LLC-PK1 celllines. Taken together, these findings suggest that in addition to a role in mineralization of bone, tissue nonspecific alkaline phosphatase might also play a role in other cell activities, including those related to differentiation, such as cell-cell or cell-substrate interaction and proliferation.     

Characterization of an arachidonic acid-deficient (Fads1 knockout) mouse model

Arachidonic acid (20:4^Δ5,8,11,14, AA)-derived eicosanoids regulate inflammation and promote cancer development. Previous studies have targeted prostaglandin enzymes in an attempt to modulate AA metabolism. However, due to safety concerns surrounding the use of pharmaceutical agents designed to target Ptgs2 (cyclooxygenase 2) and its downstream targets, it is important to identify new targets upstream of Ptgs2. Therefore, we determined the utility of antagonizing tissue AA levels as a novel approach to suppressing AA-derived eicosanoids. Systemic disruption of the Fads1 (Δ5 desaturase) gene reciprocally altered the levels of dihomo-γ-linolenic acid (20:3^Δ8,11,14, DGLA) and AA in mouse tissues, resulting in a profound increase in 1-series-derived and a concurrent decrease in 2-series-derived prostaglandins. The lack of AA-derived eicosanoids, e.g., PGE_2, was associated with perturbed intestinal crypt proliferation, immune cell homeostasis, and a heightened sensitivity to acute inflammatory challenge. In addition, null mice failed to thrive, dying off by 12 weeks of age. Dietary supplementation with AA extended the longevity of null mice to levels comparable to wild-type mice. We propose that this new mouse model will expand our understanding of how AA and its metabolites mediate inflammation and promote malignant transformation, with the eventual goal of identifying new drug targets upstream of Ptgs2.     

The tallysomycin biosynthetic gene cluster from Streptoalloteichus hindustanus E465-94 ATCC 31158 unveiling new insights into the biosynthesis of the bleomycin family of antitumor antibiotics

The tallysomycins (TLMs) belong to the bleomycin (BLM) family of antitumor antibiotics. The BLM biosynthetic gene cluster has been cloned and characterized previously from Streptomyces verticillus ATCC 15003, but engineering BLM biosynthesis for novel analogs has been hampered by the lack of a genetic system for S. verticillus. We now report the cloning and sequencing of the TLM biosynthetic gene cluster from Streptoalloteichus hindustanus E465-94 ATCC 31158 and the development of a genetic system for S. hindustanus, demonstrating the feasibility to manipulate TLM biosynthesis in S. hindustanus by gene inactivation and mutant complementation. Sequence analysis of the cloned 80.2 kb region revealed 40 open reading frames (ORFs), 30 of which were assigned to the TLM biosynthetic gene cluster. The TLM gene cluster consists of nonribosomal peptide synthetase (NRPS) genes encoding nine NRPS modules, a polyketide synthase (PKS) gene encoding one PKS module, genes encoding seven enzymes for deoxysugar biosynthesis and attachment, as well as genes encoding other biosynthesis, resistance, and regulatory proteins. The involvement of the cloned gene cluster in TLM biosynthesis was confirmed by inactivating the tlmE glycosyltransferase gene to generate a TLM non-producing mutant and by restoring TLM production to the DeltatlmE::ermE mutant strain upon expressing a functional copy of tlmE. The TLM gene cluster is highly homologous to the BLM cluster, with 25 of the 30 ORFs identified within the two clusters exhibiting striking similarities. The structural similarities and differences between TLM and BLM were reflected remarkably well by the genes and their organization in their respective biosynthetic gene clusters.     

Comparison of the neuromuscular systems among actinotroch larvae: systematic and evolutionary implications

A comparative analysis of the larval and presumptive juvenile neuromuscular systems among actinotroch larvae was performed using confocal laser microscopy with probes for F-actin and serotonin. Currently, there are two main categories of larval nervous systems based on the origin of the nerve fibers that innervate the larval tentacles. Characteristics of the serotonergic cells of the larval apical ganglion and juvenile nervous system have remained relatively conserved, but the structure of the secondary (hood) sense organ and the juvenile tentacles has diversified among species. Differences in larval musculature are mainly associated with differences in hood morphology. The presumptive, juvenile neuromuscular system is either integrated or separated from that of the larva based on the origin of the juvenile tentacles. Among species, the juvenile tentacles are made by remodeling the larval tentacles, developed from a basal tentacular thickening, or developed as a completely separate set in the larva. Differentiation of the neuromuscular structures of the juvenile tentacles is more diverse than their outward morphological characteristics would suggest. Importance of these larval characters is discussed in terms of current problems that exist within phoronid systematics. Evolutionary implications of these morphological characters are discussed among the phoronids, brachiopods, and related bilaterians. Overall, the integration or separation of larval and juvenile neuromuscular characters may yield insights into the evolution of lophotrochozoan body plans.     

Factors affecting 'Hass' avocado fruit size: Carbohydrate, abscisic acid and isoprenoid metabolism in normal and phenotypically small fruit

Carbohydrate and abscisic acid (ABA) metabolism were investigated in normal and phenotypically small 'Hass' avocado ( Persea americana Mill.) fruit in an attempt to link alterations in sugar and ABA content with changes in 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC 1.1.1.34) activity and fruit size. The small-fruit phenotype was characterized by reduced seed HMGR activity, increased seed insoluble acid invertase ( β - d -fructofuranosidase, EC 3.2.1.26), decreased sucrose synthase (SS; UDP- d -glucose: d -fructose-2- α -glucosyl-transferase, EC 2.4.1.13) activity, decreased sucrose content, and increased glucose as a proportion of the total soluble sugar. Sucrose phosphate synthase (SPS; UDP- d -glucose: d -fructose 6-phosphate 2- α - d -glucosyltransferase, EC 2.4.1.14) activity was unaffected in seed but reduced in mesocarp of the small fruit. In addition, the small-fruit variant displayed enhanced respiration and both seed and mesocarp tissue showed increased ABA metabolism. Applied ABA caused an increase in insoluble acid invertase activity in seed tissue of normal fruit while mevastatin reduced HMGR activity in this tissue, caused sucrose depletion and increased the proportion of glucose from 5 to 57% of total soluble sugars. Exogenous glucose suppressed HMGR activity in seed tissue whereas in mesocarp tissue, HMGR activity was reduced to 38% of the control after 6 h but enhanced by 46% by 48 h. Glucose increased ABA biosynthesis and turnover in competent tissues. These results suggest that ABA turnover is mediated, in part, by carbohydrate content and composition which also affects HMGR activity. It is proposed that sugar and ABA signals act in concert to modulate expression and/or activity of HMGR in the control of 'Hass' avocado fruit growth and final fruit size.     

Bioanalytical application of surface‐ and tip‐enhanced Raman spectroscopy

Due to its fingerprint specificity and trace‐level sensitivity, surface‐enhanced Raman spectroscopy (SERS) is an attractive tool in bioanalytics. This review reflects the research in this highly interesting topic of the last 3–4 years. The detection of the SERS signature of biomolecules up to microorganisms and cells is introduced. Labeling using modified nanoparticles (SERS tags) is also introduced. In order to establish biomedical applications, SERS analysis is performed in complex matrices such as body fluids. Furthermore, the SERS technique is combined with other methods such as microfluidic devices for online monitoring and scanning probe microscopy (i.e. tip‐enhanced Raman spectroscopy, TERS) to investigate nanoscaled features. The present review illustrates the broad application fields of SERS and TERS in bioanalytics and shows the great potential of these methods for biomedical diagnostics.     

Conventional kinesin holoenzymes are composed of heavy and light chain homodimers

Conventional kinesin is a major microtubule-based motor protein responsible for anterograde transport of various membrane-bounded organelles (MBO) along axons. Structurally, this molecular motor protein is a tetrameric complex composed of two heavy (kinesin-1) chains and two light chain (KLC) subunits. The products of three kinesin-1 (kinesin-1A, -1B, and -1C, formerly KIF5A, -B, and -C) and two KLC (KLC1, KLC2) genes are expressed in mammalian nervous tissue, but the functional significance of this subunit heterogeneity remains unknown. In this work, we examine all possible combinations among conventional kinesin subunits in brain tissue. In sharp contrast with previous reports, immunoprecipitation experiments here demonstrate that conventional kinesin holoenzymes are formed of kinesin-1 homodimers. Similar experiments confirmed previous findings of KLC homodimerization. Additionally, no specificity was found in the interaction between kinesin-1s and KLCs, suggesting the existence of six variant forms of conventional kinesin, as defined by their gene product composition. Subcellular fractionation studies indicate that such variants associate with biochemically different MBOs and further suggest a role of kinesin-1s in the targeting of conventional kinesin holoenzymes to specific MBO cargoes. Taken together, our data address the combination of subunits that characterize endogenous conventional kinesin. Findings on the composition and subunit organization of conventional kinesin as described here provide a molecular basis for the regulation of axonal transport and delivery of selected MBOs to discrete subcellular locations.