Threshold elemental ratios of carbon and phosphorus in aquatic consumers

Inadequate supply of one or more mineral elements can slow the growth of animal consumers and alter their physiology, life history and behaviour. A key concept for understanding nutrient deficiency in animals is the threshold elemental ratio (TER), at which growth limitation switches from one element to another. We used a stoichiometric model that coupled animal bioenergetics and body elemental composition to estimate TER of carbon and phosphorus (TER_C:P) for 41 aquatic consumer taxa. We found a wide range in TER_C:P (77–3086, ratio by atoms), which was generated by interspecific differences in body C : P ratios and gross growth efficiencies of C. TER_C:P also varied among aquatic invertebrates having different feeding strategies, such that detritivores had significantly higher threshold ratios than grazers and predators. The higher TER_C:P in detritivores resulted not only from lower gross growth efficiencies of carbon but also reflected lower body P content in these consumers. Supporting previous stoichiometric theory, we found TER_C:P to be negatively correlated with the maximum growth rate of invertebrate consumers. By coupling bioenergetics and stoichiometry, this analysis revealed strong linkages among the physiology, ecology and evolution of nutritional demands for animal growth.     

The GrlR-GrlA regulatory system coordinately controls the expression of flagellar and LEE-encoded type III protein secretion systems in enterohemorrhagic Escherichia coli

The gene function of the locus of enterocyte effacement (LEE) is essential for full virulence of enterohemorrhagic Escherichia coli (EHEC). Strict control of LEE gene expression is mediated by the coordinated activities of several regulatory elements. We previously reported that the ClpX/ClpP protease positively controls LEE expression by down-regulating intracellular levels of GrlR, a negative regulator of LEE gene expression. We further revealed that the negative effect of GrlR on LEE expression was mediated through GrlA, a positive regulator of LEE expression. In this study, we found that the FliC protein, a major component of flagellar filament, was overproduced in clpXP mutant EHEC, as previously reported for Salmonella. We further found that FliC expression was reduced in a clpXP grlR double mutant. To determine the mediators of this phenotype, FliC protein levels in wild-type, grlR, grlA, and grlR grlA strains were compared. Steady-state levels of FliC protein were reduced only in the grlR mutant, suggesting that positive regulation of FliC expression by GrlR is mediated by GrlA. Correspondingly, cell motility was also reduced in the grlR mutant, but not in the grlA or grlR grlA mutant. Because overexpression of grlA from a multicopy plasmid strongly represses the FliC level, as well as cell motility, we conclude that GrlA acts as a negative regulator of flagellar-gene expression. The fact that an EHEC strain constitutively expressing FlhD/FlhC cannot adhere to HeLa cells leads us to hypothesize that GrlA-dependent repression of the flagellar regulon is important for efficient cell adhesion of EHEC to host cells.     

Pronuclear injection-based mouse targeted transgenesis for reproducible and highly efficient transgene expression

Mouse transgenesis has proven invaluable for analysis of gene function and generation of human disease models. We describe here the development of a pronuclear injection-based targeted transgenesis (PITT) system, involving site-specific integration in fertilized eggs. The system was applied to two different genomic target loci to generate a series of transgenic lines including fluorescent mice, which reproducibly displayed strong, ubiquitous and stable transgene expression. We also demonstrated that knockdown mice could be readily generated by PITT by taking advantage of the reproducible and highly efficient expression system. The PITT system, which circumvents the problem of unpredictable and unstable transgene expression of conventional random-integration transgenic mice, reduces the time, cost and effort needed to generate transgenic mice, and is potentially applicable to both in vivo 'gain-of-function' and 'loss-of-function' studies.     

“Wisdom of the Elders” or “Loss of Experience” as a Mechanism to Explain the Decline in Traditional Ecological Knowledge: A Case Study on Awaji Island,Japan

Human Ecology - Previous studies have reported that older people are more knowledgeable about nature than the younger generation. The relationship between people’s age and level of knowledge...     

Efficient collection and cryopreservation of embryos in F344 strain inbred rats

In rats, it is now possible to produce genetically engineered strains, not only as transgenic animals but also using gene knockout techniques. Reproductive technologies have been used as indispensable tools to produce and maintain these novel valuable strains. Although studies for collecting and cryopreserving embryos have been reported using outbred rats, efficient methods have not been established in inbred strains. The F344 inbred strain is important in rat breeding and has been used for the production of transgenic/knockout strains and for genome sequencing. Here we studied the optimal conditions for oocyte collection by induction of superovulation, and the development of embryos after cryopreservation in F344 rats. The response to pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) was examined by injection of 150 IU/kg PMSG + 75 IU/kg hCG or 300 IU/kg PMSG + 300 IU/kg hCG. Superovulation was achieved at high efficiency by an injection of 150 IU/kg PMSG + 75 IU/kg hCG. Furthermore, superovulation in this strain showed similar high response as Wistar rats. Of 2-cell embryos cryopreserved by vitrification in a solution containing 10% propylene glycol, 30% ethylene glycol, 20% Percoll and 0.3 M sucrose, more than 90% survived after warming and 32% developed to offspring. However, the freezability of pronuclear stage embryos was extremely low. This study demonstrated that sufficient unfertilized oocytes and embryos can be collected from F344 rats by the induction of superovulation with 150 IU/kg PMSG + 75 IU/kg hCG. Furthermore, cryopreservation of 2-cell embryos using this vitrification protocol can now be applied to maintaining valuable rat strains derived from the F344 inbred strain as genetic resources.     

A nearly complete skeleton of an early juvenile diplodocid (Dinosauria: Sauropoda) from the Lower Morrison Formation (Late Jurassic) of north central Wyoming and its implications for early ontogeny and pneumaticity in sauropods

A nearly complete skeleton of a juvenile sauropod from the Lower Morrison Formation (Late Jurassic, Kimmeridgian) of the Howe Ranch in Bighorn County, Wyoming is described. The specimen consists of articulated mid-cervical to mid-caudal vertebrae and most appendicular bones, but cranial and mandibular elements are missing. The shoulder height is approximately 67 cm, and the total body length is estimated to be less than 200 cm. Besides the body size, the following morphological features indicate that this specimen is an early juvenile; (1) unfused centra and neural arches in presacral, sacral and first to ninth caudal vertebrae, (2) unfused coracoid and scapula, (3) open coracoid foramen, and (4) relatively smooth articular surfaces on the limb, wrist, and ankle bones. A large scapula, short neck and tail and elongate forelimb bones relative to overall body size demonstrate relative growth. A thin-section of the mid-shaft of a femur shows a lack of annual growth lines, indicating an early juvenile individual possibly younger than a few years old. Pneumatic structures in the vertebral column of the specimen SMA 0009 show that pneumatisation of the postcranial skeleton had already started in this individual, giving new insights in the early ontogenetic development of vertebral pneumaticity in sauropods.

The specimen exhibits a number of diplodocid features (e.g., very elongate slender scapular blade with a gradually dorsoventrally expanded distal end, a total of nine dorsal vertebrae, presence of the posterior centroparapophyseal lamina in the posterior dorsal vertebrae). Although a few diplodocid taxa, Diplodocus, cf. Apatosaurus, and cf. Barosaurus, are known from several fossil sites near the Howe Ranch, identification of this specimen, even at a generic level, is difficult due to a large degree of ontogenetic variation.     

Design and synthesis of new tripeptide-type SARS-CoV 3CL protease inhibitors containing an electrophilic arylketone moiety

We describe here the design, synthesis and biological evaluation of a series of molecules toward the development of novel peptidomimetic inhibitors of SARS-CoV 3CL^pro. A docking study involving binding between the initial lead compound 1 and the SARS-CoV 3CL^pro motivated the replacement of a thiazole with a benzothiazole unit as a warhead moiety at the P1′ site. This modification led to the identification of more potent derivatives, including 2i, 2k, 2m, 2o, and 2p, with IC₅₀ or K_i values in the submicromolar to nanomolar range. In particular, compounds 2i and 2p exhibited the most potent inhibitory activities, with K_i values of 4.1 and 3.1 nM, respectively. The peptidomimetic compounds identified through this process are attractive leads for the development of potential therapeutic agents against SARS. The structural requirements of the peptidomimetics with potent inhibitory activities against SARS-CoV 3CL^pro may be summarized as follows: (i) the presence of a benzothiazole warhead at the S1′-position; (ii) hydrogen bonding capabilities at the cyclic lactam of the S1-site; (iii) appropriate stereochemistry and hydrophobic moiety size at the S2-site and (iv) a unique folding conformation assumed by the phenoxyacetyl moiety at the S4-site.     

Long‐term dynamics of a cladoceran community from an early stage of lake formation in Lake Fukami‐ike,Japan

An increase in nutrient levels due to eutrophication has considerable effects on lake ecosystems. Cladocerans are intermediate consumers in lake ecosystems; thus, they are influenced by both the bottom‐up and top‐down effects that occur as eutrophication progresses. The long‐term community succession of cladocerans and the effects cladocerans experience through the various eutrophication stages have rarely been investigated from the perspective of the early‐stage cladoceran community assemblage during lake formation. In our research, long‐term cladoceran community succession was examined via paleolimnological analysis in the currently eutrophic Lake Fukami‐ike, Japan. We measured the concentration of total phosphorus and phytoplankton pigments and counted cladoceran and other invertebrate subfossils in all layers of collected sediment cores, and then assessed changes in the factors controlling the cladoceran community over a 354‐year period from lake formation to the present. The cladoceran community consisted only of benthic taxa at the time of lake formation. When rapid eutrophication occurred and phytoplankton increased, the benthic community was replaced by a pelagic community. After further eutrophication, large Daphnia and high‐order consumers became established. The statistical analysis suggested that bottom‐up effects mainly controlled the cladoceran community in the lake''s early stages, and the importance of top‐down effects increased after eutrophication occurred. Total phosphorus and phytoplankton pigments had positive effects on pelagic Bosmina, leading to the replacement of the benthic cladoceran community by the pelagic one. In contrast, the taxa established posteutrophication were affected more by predators than by nutrient levels. A decrease in planktivorous fish possibly allowed large Daphnia to establish, and the subsequent increase in planktivorous fish reduced the body size of the cladoceran community.